Proline derivatives

ABSTRACT

Proline derivatives of the formulae: ##STR1## wherein R 1  through R 11  have defined values, and acid- and base-addition salts thereof, and equilibrium addition compounds of the aldehyde group thereof; processes for their preparation; pharmaceutical compositions; and intermediates for preparing said proline derivatives. The proline derivatives are human leukocyte elastase inhibitors which are useful, for example, in treating pulmonary emphysema.

This is a division of co-pending application Ser. No. 603,408 filed onApr. 24, 1984 and now U.S. Pat. No. 4,596,789.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention comprises certain proline derivatives which areuseful as human leukocyte elastase inhibitors, e.g. in the treatment oftissue degenerative diseases such as pulmonary emphysema,atherosclerosis, rheumatoid and osteo arthritis in humans, methods fortheir use, processes used for their synthesis, intermediates useful insuch syntheses and pharmaceutical compositions prepared with suchderivatives.

2. Description of the Prior Art

Peptide aldehyde inhibitors of both porcine pancreatic elastase (PPE)and human leukocyte elastase (HLE) have been previously reported. Forexample, Thompson, Biochemistry, 1973, vol. 12, pages 47-51 hasdescribed the synthesis of two peptide aldehyde inhibitors of porcinepancreatic elastase. One of these wasN-acetyl-L-alanyl-L-prolyl-L-alaninal.

Szabo et al., Acta Biochimica et Biophysica Academiae ScientiarumHungaricae, 1982, vol. 17 (1-2), page 98, reports the synthesis of fivepeptide aldehyde compounds, all of which proved to be inhibitors forpancreatic elastase and human leukocyte elastase. The most potent HLEinhibitor reported was N-succinyl-D-phenylalanyl-L-prolyl-L-valinal;this compound produced 10-100 times stronger inhibition (K_(i) =4×10⁻⁵)than the other four aldehydes.

Although various peptide aldehydes have been reported in the literature,none has been found to be clinically useful in the treatment of anytissue degenerative disease to date.

It is generally accepted that proteolysis of lung elastin by elastases,such as human leukocyte elastase (HLE) and cathespin G, which arereleased from the granular fraction of polymorphonuclear leukocytes, isresponsible for a major part of the tissue degradation seen in pulmonaryemphysema. It is also believed that elastases are similarly involved inthe initiation and progression of various other tissue degenerativediseases, such as atherosclerosis, osteo-arthritis and rheumatoidarthritis. Accordingly, the potent elastase inhibitors of this inventionmay be used in the treatment and/or prevention of any one or more of theabove mentioned diseases. They may also be used as research tools inpharmacological and related studies.

SUMMARY OF THE INVENTION

The proline derivatives of the invention are of the following formulas(I), (II) or (III): ##STR2## wherein

R¹, R⁵ and R⁹ are lower alkyl groups containing from 3 to 6 carbonatoms;

R², R³, R⁶, R⁷ and R¹⁰ are alkyl groups of 1 to 10 carbon atoms whichmay optionally be substituted by a monocyclic aryl group or by an amide,urea or carbamate group via the nitrogen thereof;

R⁴ and R¹¹ are lower alkyl, substituted lower alkyl, lower alkoxy orsubstituted lower alkoxy groups wherein the alkyl or alkoxy contains 1to 6 carbon atoms, or monocyclic or bicyclic aryl groups; and

R⁸ is hydroxy, a lower alkoxy group containing 1 to 6 carbon atoms, oran aralkoxy group containing 7 to 12 carbon atoms;

and wherein --CHR² --, --CHR³ --, --CHR⁶ --, --CHR⁷ --, --CHR¹⁰ -- andthe proline group are of the L-configuration; or apharmaceutically-acceptable acid- or base-addition salt thereof or anequilibrium addition compound of the aldehyde group thereof.

The compounds of this invention of the formulas (I), (II) and (III) arehighly potent, reversible, selective, competitive inhibitors of humanleukocyte elastase (HLE). These compounds are up to 40,000 times morepotent than the most potent aldehyde HLE inhibitors described in theprior art; see Szabo et al. cited above in this regard, and are usefulin the treatment and/or prevention of tissue-degenerative diseases suchas emphysema, atherosclerosis, osteo-arthritis and rheumatoid arthritis.

Also part of the present invention are processes for preparing compoundsof the formula (I), (II) or (III); pharmaceutical compositionscontaining one or more compounds of the formula (I), (II) or (III) and apharmaceutically acceptable diluent or carrier; methods for thetreatment of pulmonary emphysema, atherosclerosis or osteo- orrheumatoid arthritis in a warm-blooded animal in need of such treatmentwhich comprises administering to said animal a pharmaceuticallyeffective amount of such composition; and intermediates used in thesynthesis of compounds of the formula (I), (II) or (III).

DETAILED DESCRIPTION

The compounds of the formulas (I) and (II) are tetrapeptide aldehydes,and the compounds of the formula (III) are tripeptide aldehydes.

R¹, R⁵ and R⁹ may be straight or branched chain lower alkyl of 3 to 6carbon atoms. Specific examples include n-propyl, iso-propyl, sec-butyland iso-butyl. Preferred groups R¹, R⁵ and R⁹ are alpha branched chainlower alkyl groups of 3 or 4 carbon atoms such as isopropyl andsec-butyl.

R², R³, R⁶, R⁷ and R¹⁰ may be straight or branched chain alkyl groups of1 to 10 carbons, e.g., 3 to 6 carbons, which may be substituted, e.g.,at the terminal of a straight chain alkyl, by a monocyclic aryl group orby an amide, urea or carbamate group via the nitrogen atom thereof.Examples include n-propyl, iso-propyl, n-butyl, sec-butyl or isobutylwith substitutions including benzyl, benzyloxycarbonylamino,phenylaminocarbonylamino and pyridylcarbonylamino, e.g.,2-pyridylcarbonylamino.

Preferred groups R², R⁶ and R¹⁰ are alpha branched chain lower alkylradicals of 3 to 6 carbon atoms such as isopropyl or sec-butyl, or anaralkyl radical having 7 to 12 carbon atoms such as benzyl.

Preferred groups R³ are (a) straight or branched chain lower alkylradicals of 3 to 6 carbon atoms such as n-propyl, isopropyl, n-butyl,sec-butyl or isobutyl; (b) aralkyl radicals of 7 to 12 carbon atoms suchas benzyl; or (c) straight chain alkyl groups of 1 to 8 carbon atomswhich are substituted by an amide, carbamate or urea group via thenitrogen atom thereof. Substituted alkyl radicals (c) may be representedby the formula ##STR3## where Alk is a lower alkylene radical of 1 toabout 8 carbon atoms, Y is a direct link, --O-- or --NH-- (correspondingto amide, carbamate and urea substitutents, respectively), and R¹³ islower alkyl, aryl, aralkyl, or pyridyl. Examples of substituted alkylradicals (c) include benzyloxycarbonylamino, phenylaminocarbonylamino(i.e., phenylureido), and 2-pyridylcarbonylamino.

Either R² or R³ (and similarly R⁶ and R⁷) but preferably not both, canbe amide-, carbamate-, or urea-substituted lower alkyl.

R⁴ and R¹¹ may be: (a) lower alkyl such as methyl or tert.-butyl; (b)substituted lower alkyl, such as 2-carboxyethyl, 2-methoxycarbonylethyl,acetamidomethyl, and 2-methanesulfonamidocarbonylethyl; (c) loweralkoxy, such as tert.-butoxy; (d) substituted lower alkoxy such asbenzyloxy, 2-(2-pyridyl)ethyloxy, and 2-methanesulfonylethyloxy; and (e)aryl, such as phenyl, 2,4-dichlorophenyl, 2-carboxyphenyl, and4-biphenylyl. The lower alkyl or lower alkoxy groups may have 1 to 6carbon atoms, and substituted lower alkyl or lower alkoxy groups mayhave 1 to 6 carbon atoms exclusive of those in the substituents.

R⁸ may be hydroxyl, lower alkoxy of 1 to 6 carbon atoms, or aralkoxy of7 to 12 carbon atoms.

The preferred substituents R⁸ in formula (II) compounds is lower alkoxy,such as methoxy.

The preferred substituent R¹¹ in formula (III) compounds is substitutedlower alkyl, in particular carboxyalkyl such as 2-carboxyethyl, oralkyloxycarbonylalkyl such as methoxycarbonylethyl or alkoxy such astert.-butoxy, or aralkoxy such as benzyloxy.

The equilibrium addition compounds of the aldehyde group of formulae(I), (II) or (III) include the bisulfites, hemiacetals and hemiaminals,as well as other masked aldehydes as described in "The Chemistry of theCarbonyl Group", Vol. 2 in the series "The Chemistry of FunctionalGroups", Ed. by S. Patai, Interscience Publishers, a division of JohnWiley & Sons, New York (1970).

The bisulfite addition products are those in which the --CHO group isconverted to a --CHOHSO₃ H, e.g., by reaction with a sulfite, e.g.,sodium hydrogen sulfite of the formula NaHSO₃. The reaction may beconducted by dissolving the aldehyde in a solvent such as aqueousmethanol, adding an excess, e.g., a double molar amount, of sodiumhydrogen sulfite, and removing the solvent.

The hemiacetals are formed by addition of one mole of a monohydricalcohol such as dodecanol to the --CHO group.

The hemiaminals are formed by addition of one mole of a primary aminesuch as lysine, or a secondary amine such as sarcosine (which isN-methylglycine) to the --CHO group. Addition may be carried out bydissolving the aldehyde in a suitable solvent such as methanol, addingthe amine, and removing the solvent.

The salts of the compounds of formula (I), (II) and (III) includepharmaceutically-acceptable base- or acid-addition salts such as with amineral acid, e.g., hydrochloric, or an organic acid such as citric,maleic, fumaric or acetic. Base-addition salts include those with alkalimetal hydroxides such as sodium hydroxide, alkaline earth hydroxides andorganic amine salts. Such salts may be prepared by dissolving theproline derivative in a mixture of water and a water-miscible organicsolvent, adding an aqueous solution of the base and recovering the saltin the aqueous solution.

The compounds of the present invention are of the L-configuration at allchiral centers except optionally the point of attachment of the R¹, R⁵or R⁹ group. The configuration at R¹, R⁵ and R⁹ can be D, L, or amixture thereof. Preferably the compounds of this invention are of theL-configuration at all chiral centers including the point of attachmentof the R¹, R⁵ or R⁹ group. The L-configuration at the point ofattachment of R², R⁶ or R¹⁰ is particularly important, since thecompounds of this invention, which have this configuration, are far moreactive (approximately 100 times more active) than the isomers having theD-configuration at this location. The term, "L-configuration" hereindenotes a steric configuration which is the same as that in the closestnaturally occurring L-amino acids.

Compounds of the formula (I) and their pharmaceutically-acceptable acid-and base-addition salts and aldehyde adducts constitute a preferredgroup of compounds. Especially desirable are formula (I) compoundshaving the L-configuration at all chiral centers.

Preferred compounds, all of which are of the formula (I), and of theL-configuration at all chiral centers, are the following:

(a)N-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-propyl-L-valinal,

(b)N-acetylglycyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal,

(c) N-benzyloxycarbonyl-L-norleucyl-L-valyl-L-prolyl-L-valinal,

(d)N-alpha-[3-(methylsulfonylaminocarbonyl)-propionyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal,

(e)N-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolyl-L-valinal,

(f)N-alpha-(2,4-dichlorobenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal,

(g)N-alpha-glutaryl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal,

(h)N-alpha-succinyl-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-prolyl-L-valinal,and

(i)N-alpha-(4-phenylbenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal.

and the pharmaceutically-acceptable acid- and base-addition saltsthereof and the pharmaceutically-acceptable equilibrium additioncompounds of the aldehyde group thereof. Compounds (b) through (f) areespecially preferred, and compound (a) is the most highly preferred.

The aldehydes of the formula (I), (II) or (III) can be prepared byoxidation of the corresponding alcohol or by hydrolysis ortransacetalization of the corresponding acetal (or more broadly, byremoval of an aldehyde protecting group from an aldehyde derivativehaving such group). The alcohols and acetals (or other derivativeshaving an aldehyde protecting group) in turn can be prepared accordingto peptide synthesis techniques which are known in the art.

The alcohols are compounds of the following formulas (I-A), (II-A) and(III-A): ##STR4## when R¹ to R¹¹ are as previously defined.

Aldehyde derivatives of the compounds of formulas (I), (II) and (III) inwhich the --CHO group is replaced by an aldehyde protecting group arecompounds of the following formulas (I-B), (II-B), and (III-B): ##STR5##where Q is an aldehyde protecting group and R¹ to R¹¹ are as previouslydefined. The preferred aldehyde protecting group Q is the acetal, inwhich case Q is (OR¹²)₂ where R¹² is lower alkyl of 1 to 6 carbon atomsor (in the case of an acetal formed from a diol such as ethylene glycolor propylene glycol) the two groups R¹² together with the adjacentoxygens and the carbon to which they are attached form a 5- or 6-memberheterocyclic (i.e., alkylenedioxy) ring. Alternatively, the protectinggroup may be a thioacetal, in which case Q is (SR¹²)₂ where R¹² has themeanings stated above.

The acetals are compounds of the following formulas (I-C), (II-C) and(III-C): ##STR6## where R¹ to R¹² are as previously defined.

Compounds of the invention of formulas (I), (II) and (III) can beprepared by oxidation of the corresponding alcohol of the formula (I-A),(II-A) or (III-A). This oxidation can be accomplished by any of severalprocedures including:

(a) Swern modification of a Moffatt oxidation in which the alcohol isreacted at low temperature (-78° to -20° C.) with a reagent prepared insitu by reaction of dimethyl sulfoxide (DMSO) with an activating agentsuch as oxalyl chloride or trifluoroacetic anhydride at low temperaturein a solvent such as dichloromethane (CH₂ Cl₂). This procedure isdescribed, for example, by A. J. Mancuso et al. in the Journal ofOrganic Chemistry, Vol. 43, No. 12, pages 2480-2482 (1978) and Vol. 44,No. 23, pages 4148-4150 (1979).

(b) a Collins oxidation in which the alcohol is reacted with a mixtureof chromium trioxide (CrO₃) and pyridine at low temperature (-40° to-20° C.) in a solvent such as dichloromethane.

(c) oxidation by other chromium reagents such as pyridiniumchlorochromate at temperatures from -20° C. to 25° C. in solvents suchas dichloromethane.

Alternatively, compounds of the invention of the formulas (I), (II) and(III) can be prepared by removing the protecting groups Q from acorresponding protected compound of the formula (I-B), (II-B) or(III-B). Thus, for example, compounds of the invention of formulas (I),(II) and (III) can be prepared by hydrolysis of the corresponding acetalof the formula (I-C), (II-C) or (III-C). This hydrolysis may be carriedout in the presence of an acid in a mixed aqueous-organic solventmedium. The medium may be a mixture of water and one or morewater-miscible organic solvents such as methanol, tetrahydrofuran (THF),or acetone. The medium may be acidified with an acid such ashydrochloric acid, p-toluene-sulfonic acid or a strongly acidic ionexchange resin such as Dowex® 50W×8-H (Dowex® is a registered trademarkof The Dow Chemical Company, Midland, Mich., U.S.A.). Alternatively, theacetal can be removed in a trans-acetalization reaction in the presenceof excess acetone or other low molecular weight ketones in the presenceof an acid catalyst such as p-toluene-sulfonic acid or a strongly acidicion exchange resin such as Dowex® 50W×8-H.

Compounds of the formulas (I-A), (II-A), (III-A), (I-B), (II-B),(III-B), (I-C), (II-C) and (III-C) are novel intermediates.

Both the alcohols of formulas (I-A), (II-A) and (III-A) and the aldehydederivatives (e.g., acetals) of formulas (I-B), (II-B) and (III-B) can bemade from readily available materials by peptide synthesis methods whichare known in the art. Basically, these consist of peptide coupling andprotection and deprotection reactions (to protect and deprotect aminoand carboxy groups), arranged in a sequence which will give the desiredpeptide compound.

Peptide coupling may be represented generally by equation (1) below:##STR7## where R¹⁴ and R¹⁵ are amino acid or peptide residues. In otherwords, both (V) and (VI) represent amino acids or peptides. As appliedto the present invention, the maximum number of peptide units incompound (VII) is either 3 or 4.

These peptide coupling procedures can be carried out by any one ofseveral procedures which are known in the art.

For example, coupling can be accomplished in the presence ofdicyclohexylcarbodiimide or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and1-hydroxybenzotriazole and a base such as triethylamine orN-methylmorpholine in a solvent such as tetrahydrofuran ordimethylformamide. Preferably the coupling is carried out at atemperature of from 0° to room temperature.

Alternatively, another example of the coupling procedure is the reactionof Equation (1) in which the carboxylic acid reactant is first activatedby formation of a mixed anhydride, for example a mixed carbonicanhydride. This anhydride can be prepared by reacting a carboxylic acidwith, for example, isobutyl chloroformate or ethyl chloroformate in thepresence of an amine such as triethylamine or N-methylmorpholine in asolvent such as tetrahydrofuran or dichloromethane at a temperature from-20° to 0° C. The mixed anhydride is then reacted with the aminecomponent of Equation (1) in the same solvent in the presence of thesame amine at temperatures from -40° C. to room temperature to providethe coupled product.

In some cases the acid (V) of equation (1) can be reacted in the form ofits acid halide or acid anhydride derivative with the amine (VI) in thepresence of a tertiary amine such a N-methylmorpholine or triethylaminein solvents such as tetrahydrofuran or dichloromethane at a temperatureof from -20° C. to room temperature. Alternatively, the acid chloridecan be reacted with the amine in an aqueous solution containing sodiumhydroxide. In some instances a water-immiscible solvent such as ether ordichloromethane is present. The reaction is conducted at temperatures of-10° C. at room temperature.

Other free amino groups or free carboxylic acid groups in the reactingcompounds (V) and (VI) must sometimes be temporarily protected duringthe coupling reaction for the coupling to proceed in the desired manner.These protecting groups, methods for their introduction, and methods fortheir removal are known in the art. The protecting groups are chosen sothat they may be selectively introduced and removed under conditionswhich do not affect other reactive groups in the compounds.

Examples of amino protecting groups are tert-butyloxycarbonyl, which maybe removed by anhydrous acid such as trifluoroacetic acid or hydrogenchloride in a solvent such as ether, ethyl acetate or dichloromethane;benzyloxycarbonyl, which may be removed by catalytic hydrogenolysis inthe presence of a catalyst such as palladium on carbon in a solvent suchas ethanol or ethyl acetate; and methylsulfonylethyloxycarbonyl, whichmay be removed by treatment with a base such as sodium hydroxide in asolvent such as aqueous methanol or tetrahydrofuran.

Acid protecting groups include the methyl ester, which may be removed bybasic hydrolysis in the presence of sodium hydroxide or sodium carbonatein a solvent such as aqueous tetrahydrofuran or methanol; the tert-butylester, which may be removed in the presence of acids such as hydrogenchloride or trifluoroacetic acid in a solvent such as ether, ethylacetate or dichloromethane; and the benzyl ester, which may be removedby basic hydrolysis as above or by catalytic hydrogenolysis as describedabove. These protecting groups can be removed immediately after acoupling reaction or left in place until a later stage in the synthesisand removed at that time.

Various methods can be used to prepare alcohols of the formula (I-A).The following are illustrative.

A first method for preparing alcohols of formula (I-A) involves reactingcompounds of the following formulae (VIII) and (IX) to produce (X):##STR8## wherein R¹⁶ and R¹⁷ are arylalkyl or alkyl groups, e.g.,benzyl, methyl or tert-butyl. For example, the coupling is conducted inthe presence of coupling promoters such as dicyclohexylcarbodiimide(DCC) and 1-hydroxybenzotriazole (HOBT) or by the mixed anhydrideprocedure with isobutyl chloroformate. Unless otherwise indicated, thedefinitions of the various R groups throughout the specification are asdefined previously. The --COOR¹⁶ protecting group is then removed bycatalytic hydrogenolysis or acid-catalyzed deprotection withconcentrated mineral acid or a strong organic acid such astrifluoroacetic acid, and the free base or salt is reacted with an acidof the formula (XI) to produce a compound of formula (XII): ##STR9## atabout 0° C. to room temperature. From the ester of formula (XII), theOR¹⁷ group is replaced by --OH by saponification with NaOH at about roomtemperature or via catalytic hydrogenolysis and this carboxylic acid isreacted with an aminoalcohol of the formula (XIII) to produce thecompound of the following formula (I-A): at a temperature of about -40°to -23° C. In the coupling reaction to produce (I-A), isobutylchloroformate is used to produce a mixed carbonic anhydride of thecarboxylic acid of the ester (XII) in the presence ofN-methylmorpholine. Compounds of the formula (XIII) may be produced fromthe corresponding aminoacid of the formula H₂ N--CHR¹ --COOH by firstesterifying the acid with an alcohol such as methanol or ethanol, toproduce the aminoester of the formula H₂ N--CHR¹ --COOR¹⁸ where R¹⁸ isan alkyl group of 1 to 2 carbons, and reducing the aminoester with areducing agent such as lithium aluminum hydride to yield (XIII), H₂N--CHR⁵ --CH₂ OH or H₂ N--CHR⁹ --CH₂ OH. The alcohol of formula (I-A)may be oxidized to produce a compound of formula (I) as described above.

A second method for preparing formula (I-A) compounds involves reactingthe formula (XI) acid with an amine of formula (XIV) to produce acompound of formula (XV): ##STR10## wherein R¹⁹ is an alkyl or arylalkylgroup, e.g. methyl, at a temperature of about -40° to -20° C., e.g.,using N-methyl-morpholine and isobutyl chloroformate as describedpreviously. The formula (XV) ester is then saponified by reaction withan alkali metal hydroxide at about room temperature and the acid isreacted with a compound of formula (IX) to yield formula (XII) which maythen be taken on to (I-A) and (I) as described above.

A third method of preparing formula (I-A) compounds involves changingthe substitution on the R² or R³ alkyl groups. It should be noted thatsuch change may also take place in preparing compounds of the inventionof formulas (II-A) and (III-A). Thus, a compound of formula (XII)wherein at least one of R² or R³ is substituted alkyl may be reacted togive the corresponding amine of the formula (XVI): ##STR11## wherein atleast one of R²⁰ and R²¹ is an alkyl group of 1 to 10 carbonssubstituted by a --NH₂ under conditions as described herein for theremoval of a --COOR¹⁶ group, in particular if the substitution is acarbamate. The amine (XVI) may then be either reacted with a carboxylicacid to produce a compound of formula (XII) wherein the R² and/or R³which had been alkyl substituted by carbamate is transformed into analkyl substituted by an amide, or reacted with an isocyanate to producea compound of formula (XII) wherein the R² and/or R³ which had beenalkyl substituted by carbamate is transformed into an alkyl substitutedby a urea moiety. Both of such formula (XII) compounds may be saponifiedand then condensed with a compound of formula (XIII) to produce compound(I-A).

In some instances the synthetic methods used to produce compounds (I),(II) and (III) involve substitution of one R⁴, R⁸ or R¹¹ group foranother in compounds (I-A), (II-A) and (III-A). For example, when it isdesired to substitute a carboxyalkyl group for a tert-butoxy group incompounds of formulas (I-A) and (III-A), the tert-butoxy group isremoved by acid catalyzed deprotection, e.g., with trifluoroacetic acid,and the resulting amine is reacted with a monoester-acid chloride oranhydride such as succinic, glutaric or adipic anhydride (e.g., at roomtemperature in acetonitrile) to produce a second compound of the formula(I-A) or (III-A), in which R⁴ or R¹¹ is carboxyalkyl (e.g.,carboxyethyl), alkoxycarbonylalkyl, aryloxycarbonylalkyl, oraralkoxycarbonylalkyl. The second compound (I-A), (II-A) or (III-A) maythen be oxidized to the corresponding compound (I), (II) or (III).

Compounds of the formula (II-A) may be produced by preparing a compoundof the formula (X) from compounds of fomulas (VIII) and (IX) asexplained above, removing the --COOR¹⁶ protecting group and reacting thefree base or salt with an isocyanate of the formula (XVII) to producethe urea of the following formula (XVIII): ##STR12## by reaction at 0°C. to room temperature. The isocyanate of formula (XVII) may be preparedfrom the corresponding aminoacid ester of the formula R⁸ --CO--CHR⁷--NH₃ ⁺ Cl⁻ by reaction with phosgene at a temperature of 115° C. Theurea (XVIII) is then converted to the corresponding carboxylic acid,replacing OR¹⁷ where R¹⁷ is benzyl with OH by catalytic hydrogenolysiswith a noble metal as the catalyst and hydrogen. The thus-producedcarboxylic acid is then condensed with an aminoalcohol of the formula H₂N--CHR⁵ --CH₂ OH, as described above for the reaction of compound (XIII)to produce (I-A), to produce the compound of the formula (II-A). Thecompound of formula (II-A) is then oxidized to the desired product offormula (II) as described above.

Compounds of the formula (III-A) may be produced by condensing acompound of the formula (XI), wherein R³ and R⁴ are values of R¹⁰ andR¹¹, respectively, with a compound of the formula (IX) to yield acompound of the following formula (XIX): ##STR13## with DCC and HOBT asdescribed for the coupling of (VIII) and (IX) to produce (X). The esterof formula (XIX) may then be cleaved by saponification or hydrogenolysisas described above for the reaction of (XII) to yield the correspondingacid. The acid is then condensed with an aminoalcohol of the formula H₂N--CHR⁹ --CH₂ OH, as described above for the preparation of compounds(I-A) from (XII), to produce the compound of the formula (III-A). Thecompound of formula (III-A) is then oxidized to the desired product offormula (III) as described above.

Compounds of the formulas (I-B), (II-B) and (III-B) can also be preparedaccording to known peptide synthesis techniques. A preferred method forpreparing acetals of the formula (I-C) will now be described forpurposes of illustration.

According to a preferred method of preparing an acetal of the formula(I-C), a compound of the formula (VIII) is reacted with a compound (IX)to form a compound (X) as previously described. In particular, R¹⁶ informulas (VIII) and (X) may be benzyl. The --COOR¹⁶ protecting group isremoved, e.g., by catalytic hydrogenolysis, as described previously. Theresulting free amine or salt is then reacted with a compound of theformula (XI) under known coupling conditions, e.g., at 0° C. at roomtemperature in the presence of coupling promoters such as DCC and HOBT,to produce a compound of the formula (XII). Preferably in formula (XII),R⁴ is tertbutyloxy and R¹⁷ is tert-butyl. In this case, reaction of(XII) with trifluoroacetic acid results in replacement of --OR¹⁷ with--OH and removal of R⁴ CO--. The resulting amine or acid addition saltis reacted with a suitable acylating agent containing R⁴, e.g., amonoester-acid chloride of succinic acid, giving a compound of thefollowing formula (XX): ##STR14## where R⁴ is, for example,carboxyalkyl, alkyloxycarbonylalkyl or aralkyloxycarbonylalkyl.

An aminoalcohol of the formula (XIII) (e.g., L-valinol) is coupled withbenzyl chloroformate at 0° to 25° C. to protect the amino group. Theamino-protected derivative of (XIII) is then oxidized by means describedearlier in this specification, e.g., by a Swern modification of aMoffatt oxidation, to give an aldehyde of the following formula (XXI):

    R.sup.22 OOC--NH--CHR.sup.1 --CHO                          (XXI)

where R²² is lower alkyl or aralkyl, e.g., benzyl. Acid-catalyzedacetalization, e.g., with triethyl orthoformate in absolute alcoholacidified with p-toluenesulfonic acid at room temperature, yields anacetal (e.g., the diethyl acetal) of (XXI). The amine protecting groupmay be removed, e.g., by catalytic hydrogenolysis when R²² is benzyl, togive an amino-acetal of the following formula (XXII):

    H.sub.2 N--CHR.sup.1 --CH(OR.sup.12).sub.2                 (XXII).

Compounds (XX) and (XXII) are coupled under suitable couplingconditions, e.g., in the presence of isobutyl chloroformate at atemperature of -40° C. to room temperature, to yield a compound of theformula (I-C). When R⁴ is hydrolyzable under basic conditions, as forexample when R⁴ is alkoxycarbonylalkyl, the R⁴ group may be converted toan alternate R⁴ group by basic hydrolysis, for example, the conversionof methoxycarbonylethyl to carboxyethyl. Hydrolysis ortransacetalization of (I-C) yields the desired compound (I).

The above synthesis is particularly useful for preparingN-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal,which is a highly preferred compound of the formula (I) of thisinvention.

Other reactants and sequences of steps can be used when it is desired toproduce other compounds of the formula (I), (II) or (III) of thisinvention via the corresponding acetals.

According to a further feature of the invention there are providedpharmaceutical compositions comprising a pharmaceutically-effectiveamount of at least one proline derivative of the formula (I), (II) or(III), wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are asdefined above, or a pharmaceutically-acceptable acid- or base-additionsalt thereof, or a pharmaceutically-acceptable equilibrium additioncompound of the aldehyde group thereof, and apharmaceutically-acceptable diluent or carrier.

The potency of compounds of the invention to act as elastase inhibitorswas determined by the ability of a compound of the invention to inhibitthe action of human leukocyte elastase (HLE) on a low molecular weightpeptide substrate.

The potency of an inhibitor is evaluated by obtaining a kineticdetermination of the dissociation constant, K_(i), of the complex formedfrom the interaction of an inhibitor with HLE. The substrate used wasthe anilide methoxysuccinyl-alanyl-alanylprolyl-valine-p-nitroanilide asdescribed by K. Nakajima et al. in The Journal of Biological Chemistry,Vol. 254, pages 4027-4032 (1979) and by T. Teshima et al. in The Journalof Biological Chemistry, Vol. 257, No. 9, pages 5085-5091 (1982). TheHLE enzyme used in these studies may be obtained according to B. R.Viscarello et al. in Preparative Biochemistry, Vol. 13 (1983).

From HLE enzyme obtained as described in the Viscarello et al reference,a standard rate of production of p-nitroaniline was measured at roomtemperature spectrophotometrically in the visible spectrum at 410nanometers, with automatic data acquisition in a Cary 210-Apple III Plusmicrocomputer system obtained from Varian Associates. Reactions wereinitiated by injection of a small aliquot (50 or 100 microliters) of astock solution of the anilide substrate in DMSO into a cuvettecontaining buffer and HLE enzyme. The amount of HLE used is sufficientto produce an initial velocity of about 0.01 OD (optical density) perminute. Final volume was 3.0 ml. Initial velocities of production ofp-nitroaniline were determined from linear regression analysis of atleast 30 data points (OD₄₁₀ vs. time) taken during the initial, linear,portion of the reaction. The initial velocity for the HLE sample withoutany inhibitor being present was obtained and used as a standard forsubsequent determination of the initial velocity of the reaction in thepresence of an inhibitor to be tested. The K_(i) value of a particularinhibitor was determined as follows. To a 3 ml cuvette were added 2.78ml of a buffer (9 millimolar sodium phosphate, 10% DMSO (v/v), pH=7.9),100 microliters of inhibitor solution in DMSO and 20 microliters of HLEstock solution at the same concentration used in control. After theinhibitor and enzyme solution had equilibrated, the reaction wasinitiated by the addition of 100 microliters of the stock substratesolution in DMSO. The final solution was 16.6% (v/v) in DMSO, with pH8.1. The substrate was at an initial concentration of 1.6×10⁻⁴ molar.Duplicate or triplicate runs were done at zero inhibitor concentrationas controls and at least three non-zero inhibitor concentrationstypically from 1×10⁻⁵ M to 1×10⁻⁸ M. A Dixon plot (reciprocal velocityvs. inhibitor concentration) was used to determine the K_(i) asdescribed by I. H. Segal in "Enzyme Kinetics", pages 109-111, John Wileyand Sons, New York (1975). Variation in the percent DMSO will result insmall changes in the K_(i) values. The K_(i) value for the product ofExample 32g was 9.4×10⁻⁸ molar; Example 1f was 1.5×10⁻⁸ molar; Example5b was 3.0×10⁻⁸ molar; and Example 8b was 6×10⁻⁹ molar. Preferredcompounds of this invention have Ki values in the range 10⁻⁹ molar to10⁻⁷ molar.

Pharmacokinetics: Male Syrian hamsters (80 to 120) g) are injectedsubcutaneously with the test compound. Prior to injection and at varyingtime periods thereafter they are lightly anesthetized with ether andblood samples of approximately 0.2 ml each are withdrawn by cardiacpuncture. The blood is expressed into 2 ml centrifuge tubes and allowedto clot for one hr. The sample is then centrifuged and the serumremoved. Blood samples are taken before (time 0) and 15, 30, 60, 90 and120 minutes after subcutaneous administration of the test compound in anappropriate vehicle.

Drug levels are determined by first inactivating endogenous elastaseinhibitors by incubation of 50 microliters of serum with an equal volumeof buffer containing 5 mg/ml bovine pancreatic trypsin for 5 min. Thetrypsin inactivated serum (40 microliters) is then added to a 3 mlcuvette containing buffer made 20 nM with respect to Human LeukocyteElastase. After an additional 2 min. incubation, the reaction is startedby the addition of substrate (100 microliters)(MeOSuc-Ala-Ala-Pro-Val-pNA, 1.6 mM) and the reaction monitoredspectophotometrically at a wavelength of 410 nm. Serum concentrations ofthe test compounds are determined by the following equation: ##EQU1##

where [I], [E] and [S] are the assay concentrations of inhibitor enzymeand substrate respectively, Ki is the inhibition constant of theinhibitor being assayed, Km is the Michaelis constant of the substrate,Vo is the control uninhibited reaction velocity, and Vi is the inhibitedreaction velocity. Velocity of the time 0 serum reaction is taken as V.Data are expressed as molar concentration of inhibitor in serum vs timepost inhibitor administration. An approximate serum half-life (t1/2) iscalculated from the curve.

The compoundN-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal(the compound of examples 5 and 6) is highly preferred because, inaddition to high potency as measured by its Ki value, this compound hasgood activity in animals as measured by the above test.

None of the invention compounds which have been tested in vivo exhibitedany signs of toxicity at the concentrations administered (about 30 mg/kgof body weight).

The compounds of the present invention may be administered to an animalparticularly a human in need thereof, for the alleviation of conditionsincluding pulmonary emphysema, atherosclerosis and osteo and rheumatoidarthritis, in particular for emphysema. The mode of administration maybe parenteral, including the subcutaneous deposit of an osmotic pump, orvia a powdered or liquid aerosol. For parenteral administration, a 1 to10 ml intravenous, intramuscular or subcutaneous injection would begiven containing about 0.02 to 10 mg/kg of body weight of a compound ofthe invention 3 or 4 times daily. The injection would contain a compoundof the invention in an aqueous isotonic sterile solution or suspensionoptionally with a preservative such as phenol or a solubilizing agentsuch as ethylenediaminetetraacetic acid (EDTA). In a powdered aerosol,compounds of the invention may be administered in the same manner ascromolyn sodium via a Spinhaler® turbo-inhaler device obtained fromFisons Corp. of Bedford, Mass. at a rate of about 0.1 to 50 mg percapsule, 1 to 8 capsules being administered daily for an average human.Each capsule to be used in the Spinhaler® contains the required amountof a compound of the invention with the remainder of the capsule being apharmaceutically-acceptable carrier such as lactose. In a liquidaerosol, the compounds of the invention are administered at the rate ofabout 100 to 1000 micrograms per "puff" or activated release of astandard volume of propellant. The liquid aerosol would be given at therate of 1 to 8 puffs per day with variation in dosages due to theseverity of the condition being treated, the weight of the patient andthe particle size distribution of the aerosol since smaller particleswill achieve greater lung penetration. Propellants, e.g., a fluorinatedhydrocarbon or isobutane, containers, valves and actuators for liquidaerosols are described by L. Lachman et al. in "The Theory and Practiceof Industrial Pharmacy", Lea and Febiger, Philadelphia (1976).

In the following Examples and throughout the specification, thefollowing abbreviations are used: atm (atmospheres); bp (boiling point);°C. (degrees Centigrade); g (grams); hr (hours); mg (milligrams); min(minutes); ml (milliliters); mmol (millimoles); mp (melting point); N(normal); nm (nanometers); nM (nanomolar); R_(f) (relative mobility inTLC); TLC (thin layer chromatography); DCC (dicyclohexylcarbodiimide);DMF (dimethylformamide); DMSO (dimethyl sulfoxide); Et₂ O (diethylether); EtOAc (ethyl acetate); HOAc (acetic acid); HOBT(hydroxybenzotriazole); MeOH (methyl alcohol); Pd/C (palladium oncharcoal catalyst); pNA (paranitroanilide); and THF (tetrahydrofuran).In addition, C, H, N, etc. (the conventional symbols for the elements)are used.

The L-configuration is indicated in the following Examples andthroughout the specification in all instances where chirality exists andthe configuration is not specified.

EXAMPLE 1N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NH-- COOCH₂C₆ H₅ ; R⁴ ═--OC(CH₃)₃.

a. N-alpha-t-Butyloxycarbonyl-L-valyl-L-proline methyl ester

1-Hydroxybenzotriazole (65.4 g, 0.484 mol) was added to a solution ofN-t-butyloxycarbonyl-L-valine (52.5 g, 0.242 mol) in 600 ml of dry DMFat 0° C. under a nitrogen atmosphere and stirred for 15 min. To theresulting solution was added a suspension of L-proline methyl esterhydrochloride (40.0 g, 0.242 mol) with TEA (36 ml, 0.25 mol) in dry DMFfollowed by DCC (55.0 g, 0.266 mol). The mixture was stirred for 3 hr.at 0° C. and then at room temperature for 4 days. The reaction mixturewas filtered and the filtrate concentrated under vacuum. The resultingresidue was mixed with 1.0 l of EtOAc and filtered. The filtrate waswashed successively with 20% aqueous citric acid, brine, saturatedaqueous NaHCO₃ and brine. The organic phase was dried over Na₂ SO₄,filtered and concentrated to give 80.7 g of the product as a thickyellow syrup; TLC, R_(f) =0.4, silica gel, EtOAc:CHCl₃ (15:85).

b. L-Valyl-L-proline methyl ester hydrochloride

A solution of 6N HCl/EtOAc (200 ml) was added to a solution of theproduct of Example 1a in EtOAc (300 ml) at 0° C.

The reaction mixture was warmed to room temperature and stirredovernight. The resulting solution was concentrated under vacuum. Theresidual solid was triturated with Et₂ O to afford 33.9 g of the productas a white solid.

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolinemethyl ester

Isobutyl chloroformate (16.7 g, 0.122 mol) was added to a solution ofN-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysine (46.4 g,0.122 mol), N-methylmorpholine (13.2 g, 0.13 mol) in dry THF (300 ml) at-25° C. under a nitrogen atmosphere. A white precipitate immediatelyformed. The reaction mixture was stirred for 1 hr. and then cooled to-60° C. A pre-cooled (-78° C.) suspension of the product of Example 1b(33.0 g, 0.122 mol) with N-methylmorpholine (13.2 g, 0.13 mol) in dryTHF (300 ml) was added in one portion. The reaction mixture was warmedslowly to room temperature and stirred overnight. The resulting mixturewas filtered and the filtrate concentrated under vacuum to give aresidue which was dissolved in EtOAc and washed successively withaqueous 1N HCl, saturated aqueous NaHCO₃ and brine. The organic phasewas dried over Na₂ SO₄, filtered and concentrated under vacuum to givethe product (82.0 g) as a pale yellow oil; TLC, R_(f) =0.65, silica gel,EtOAc.

d.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-proline

A solution of NaOH (0.75 g, 18.75 mmol) in H₂ O (10 ml) was added to astirred solution of the product of Example 1c (10.1 g) in MeOH (50 ml)at room temperature. The reaction mixture was stirred overnight and thenconcentrated under vacuum. The resulting residue was dissolved in H₂ Oand the solution extracted with Et₂ O. The aqueous layer was acidifiedwith 5% aqueous citric acid and extracted with EtOAc. The organic phasewas dried with Na₂ SO₄, filtered and concentrated under vacuum to givethe product (9.27 g) as a white solid; TLC, R_(f) =0.55, silica gel,MeOH:CHCl₃ :HOAc (9.5:90:0.5).

e.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinol

A solution of the product of Example 1d in dry THF (100 ml) under anitrogen atmosphere was cooled to -20° C. N-methylmorpholine (1.6 ml;14.8 mmol) was added followed by isobutylchloroformate (1.9 ml, 14.8mmol). The mixture was stirred for 10 min. and then cooled to -40° C. Asolution of L-valinol (1.5 g; 14.8 mmol) in dry THF (50 ml) was addeddropwise. The solution was allowed to warm slowly to room temperatureand stirred overnight. The reaction mixture was filtered, the filtrateconcentrated under vacuum and the residue dissolved in EtOAc. The EtOAcsolution was washed successively with aqueous 1N HCl, saturated aqueousNaHCO₃ and brine. The organic phase was dried with Na₂ SO₄, filtered andconcentrated in vacuum to give 9.06 g of the crude alcohol. The alcoholwas purified by flash chromatography on silica gel with MeOH:EtOAc(3:97) as the eluent to give the product; TLC, R_(f) =0.25, silica gel,MeOH:EtOAc (5:95).

Elemental Analysis. Calculated for C₃₄ H₅₅ N₅ O₈.H₂ O: C, 60.07; H,8.45; N, 10.30. Found: C, 60.00; H, 8.22; N, 10.55.

f.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A solution of DMSO (85 microliters) in dry CH₂ Cl₂ (1 ml) was addeddropwise with a syringe to a stirred solution of oxalyl chloride (52microliters, 0.6 mmol) in dry CH₂ Cl₂ (1 ml) cooled to -23° C. under anitrogen atmosphere. A solution of the product of Example 1e (0.20 g,0.3 mmol) in CH₂ Cl₂ (1.5 ml) was added in the same manner. The reactionmixture was stirred for 30 minutes and TEA (166 microliters, 1.2 mmol)added dropwise with a syringe. The reaction mixture was allowed toslowly warm to room temperature and then diluted with CH₂ Cl₂ and washedsuccessively with 10% aqueous acetic acid, 5% aqueous NaHCO₃ and brine.The organic phase was dried with Na₂ SO₄, filtered and concentratedunder vacuum to give the crude aldehyde. The aldehyde was purified usingtwo successive flash chromatographies with silica gel and eluents of CH₂Cl₂ :EtOAc (3:7) and MeOH:CHCl₃ (3:97) respectively to give the product(90 mg); TLC, R_(f) =0.45, silica gel, MeOH:Et₂ O (5:95).

Elemental Analysis. Calculated for C₃₄ H₅₃ N₅ O₈.H₂ O: C, 60.24; H,8.18; N, 10.33. Found: C, 60.01; H, 7.84; N, 10.09.

Preparation of bisulfite adducts of this and other Formula (I) aldehydesof this invention is described in Example 31.

EXAMPLE 2N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--(CH₂)₄ NHCOOCH₂ C₆ H₅ ; R³ ═--(CH₂)₄NHCOOCH₂ C₆ H₅ ; R⁴ ═--OC(CH₃)₃.

a.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysinemethyl ester

A solution of isobutyl chloroformate (0.79 g, 5.79 mmol) in CH₂ Cl₂ (10ml) was added dropwise to a stirred solution ofN-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxy-carbonyl-L-lysine (2.00g, 5.26 mmol) and N-methylmorpholine (1.33 g, 13.15 mmol) in CH₂ Cl₂ (50ml) cooled to -23° C. under a nitrogen atmosphere. The mixture wasstirred for an additional 15 min. andN-epsilon-benzyloxycarbonyl-L-lysine methyl ester hydrochloride (1.74 g,5.26 mmol) was added. The reaction mixture was allowed to warm slowly toroom temperature and stirred overnight. The mixture was washedsuccessively with 5% aqueous NaHCO₃, twice with 5% aqueous citric acid,and brine. The organic phase was dried with Na₂ SO₄, filtered andconcentrated under vacuum. The product was purified by flashchromatography on silica gel with MeOH:CHCl₃ (3:97) as the eluent togive the product (2.20 g) as a clear syrup. The compound was trituratedwith Et₂ O to give the product as a white precipitate: mp=108°-110° C.;TLC, R_(f) =0.59, silica gel, MeOH:CHCl₃ (3:97).

b.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysine

The product of Example 2a (0.12 g, 3.05 mmol) was mixed with NaOH (0.12g, 3.05 mmol) and MeOH (50 ml) was stirred overnight at roomtemperature. The solution was concentrated under vacuum at roomtemperature and the resulting residue dissolved in H₂ O (40 ml). HOAc(3.36 mmol) was added and the solid product was isolated by decantation.The product was dissolved in CH₂ Cl₂, washed successively with 5%aqueous citric acid and brine, and dried with Na₂ SO₄. The solution wasfiltered and concentrated under vacuum to give the product (1.62 g) as aclear syrup; TLC, R_(f) =0.10, silica gel, MeOH:CHCl₃ (5:95).

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolinemethyl ester

The title compound was prepared as in Example 2a, using the product ofExample 2b (1.51 g, 2.35 mmol) and N-methylmorpholine (0.59 g, 5.88mmol) in CH₂ Cl₂ (50 ml) and adding isobutyl chloroformate (0.35 g, 2.59mmol) and L-proline methyl ester hydrochloride (0.39 g, 2.35 mmol) togive the product (0.95 g); TLC, R_(f) =0.95, silica gel, MeOH:CHCl₃(3:97).

d.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-proline

The title compound was prepared as in Example 2b using the product ofExample 2c (0.95 g, 1.26 mmol), NaOH (0.56 g, 1.4 mmol) and MeOH (50ml). The product was purified by flash chromatography on silica gel withMeOH:CHCl₃ :HOAc (4.5:95:0.5) to give the product (0.75 g); TLC, R_(f)=0.50, silica gel, MeOH:CHCl₃ :HOAc (4.5:95:0.5).

e.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinol

The title compound was prepared as in Example 2a using the product ofExample 2d (0.42 g, 0.568 mmol) and N-methylmorpholine (0.06 g, 0.568mmol) in CH₂ Cl₂ (25 ml) and adding isobutyl chloroformate (0.08 g,0.568 mmol) and L-valinol (0.06 g, 0.568 mmol). The product was purifiedby flash chromatography on silica gel using MeOH:CHCl₃ (3:97) as eluentto give the title compound (0.18 g); TLC, R_(f) =0.48, silica gel,MeOH:CHCl₃ (3:97).

f.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinal

The title compound was prepared as in Example 1f using oxalyl chloride(0.03 g, 0.213 mmol) in CH₂ Cl₂ (2 ml) and adding DMSO (0.03 g, 0.380mmol) in CH₂ Cl₂ (2 ml), the product of Example 2e (0.16 g, 0.194 mmol)in CH₂ Cl₂ (2 ml), and TEA (0.10 g, 1 mmol) in CH₂ Cl₂ (2 ml). The crudemixture was separated by flash chromatography to give the product (0.022g); TLC, R_(f) =0.47, silica gel, MeOH:CHCl₃ (3:97).

Elemental Analysis. Calculated for C₄₃ H₆₂ N₆ O₁₀ : C, 62.75; H, 7.59;N, 10.21. Found: C, 62.54; H, 7.45; N, 10.41.

EXAMPLE 3N-alpha-t-Butyloxycarbonyl-N-epsilon-picolinyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄NHCO-2-pyridyl; R⁴ ═--OC(CH₃)₃.

a. N-alpha-t-Butyloxycarbonyl-L-lysyl-L-valyl-L-proline methyl ester

The product of Example 1c (5.88 g, 9.95 mmol) was mixed with 10% Pd/C(1.25 g) and MeOH (100 ml) and placed in a Parr shaker (3 atm. of H₂).The reaction was stopped after the theoretical amount of H₂ wasconsumed. The mixture was filtered through diatomaceous earth (Celite®)and concentrated under vacuum to give the product (4.46 g); TLC, R_(f)=0.10, silica gel, MeOH:CHCl₃ (3:97). (Celite® is a registered trademarkof Manville Corporation, Denver, Colo., U.S.A.).

b.N-alpha-t-Butyloxycarbonyl-N-epsilon-picolinyl-L-lysyl-L-valyl-L-prolinemethyl ester

A solution of picolinic acid (0.65 g, 5.32 mmol), HOBT (1.44 g, 10.64mmol) and N-methylmorpholine (0.54 g, 5.32 mmol) in DMF (100 ml) wascooled to 0° C. and DCC (1.21 g, 5.85 mmol) added. The mixture wasstirred for an additional 15 min. and the product of Example 3a (2.43 g,5.32 mmol) added. The reaction mixture was allowed to warm to roomtemperature and stirred overnight. The mixture was filtered andconcentrated under vacuum. The residue was dissolved in CH₂ Cl₂, washedsuccessively with H₂ O, three times with 5% aqueous NaHCO₃ and brine.The organic layer was dried over Na₂ SO₄, filtered and concentratedunder vacuum to give the crude product, which was purified by flashchromatography on silica gel with MeOH:CHCl₃ (5:95) as eluent to givethe product (1.45 g) as a tan hygroscopic foam; TLC, R_(f) =0.35, silicagel, MeOH:CHCl₃ (5:95).

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-picolinyl-L-lysyl-L-valyl-L-proline

The title compound was prepared as in Example 2b, using the product ofExample 3b (1.05 g, 1.87 mmol) and NaOH (0.11 g, 2.8 mmol) in MeOH (50ml) to give the product (0.87 g); TLC, R_(f) =0.80, silica gel,MeOH:CHCl₃ (5:95).

d.N-alpha-t-Butyloxycarbonyl-N-epsilon-picolinyl-L-lysyl-L-valyl-L-prolyl-L-valinol

The title compound was prepared as in Example 2a using the product ofExample 3c (1.17 g, 2.14 mmol) and N-methylmorpholine (0.24 g, 2.35mmol) in CH₂ Cl₂ (40 ml) and adding isobutyl chloroformate (0.29 g, 2.14mmol) in CH₂ Cl₂ (20 ml) followed by L-valinol (0.22 g, 2.14 mmol) togive the product (1.07 g) as a white foam; TLC, R_(f) =0.35, silica gel,MeOH:CHCl₃ (3:97).

e.N-alpha-t-Butyloxycarbonyl-N-epsilon-picolinyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The title compound was prepared as in Example 1f using oxalyl chloride(0.92 g, 7.25 mmol) in CH₂ Cl₂ (20 ml) and adding DMSO (1.13 g, 14.5mmol) in CH₂ Cl₂ (5 ml), the product of Example 3d (0.92 g, 1.45 mmol)in CH₂ Cl₂ (5 ml), and TEA (7.34 g, 72.5 mmol) in CH₂ Cl₂ (5 ml). Thecrude mixture was purified by flash chromatography on silica gel withMeOH:CHCl₃ (5:95) to give the product (0.23 g); TLC, R_(f) =0.66, silicagel, MeOH:CHCl₃, (5:95).

Elemental Analysis. Calculated for C₃₂ H₅₀ N₆ O₇.2H₂ O: C, 57.64; H,8.16; N, 12.61. Found: C, 57.57; H, 7.96; N, 12.51.

EXAMPLE 4N-alpha-t-Butyloxycarbonyl-N-epsilon-phenylcarbamoyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCONHC₆ H₅; R⁴ ═--OC(CH₃)₃.

a.N-alpha-t-Butyloxycarbonyl-N-epsilon-phenylcarbamoyl-L-lysyl-L-valyl-L-prolinemethyl ester

A solution of the product of Example 3a (1.0 g, 2.0 mmol) and phenylisocyanate (0.23 ml, 2.1 mmol) in CHCl₃ (15 ml) was stirred at roomtemperature for 24 hrs. The reaction mixture was concentrated undervacuum, the residue dissolved in EtOAc, and the solution washedsuccessively with aqueous 1N HCl, saturated aqueous NaHCO₃ and brine.The organic layer was dried over Na₂ SO₄, filtered and concentratedunder vacuum to give the product (1.25 g) as a white foam; TLC, R_(f)=0.36, silica gel, EtOAc.

b.N-alpha-t-Butyloxycarbonyl-N-epsilon-phenylcarbamoyl-L-lysyl-L-valyl-L-proline

The title compound was prepared as in Example 1d, using the product ofExample 4a (0.75 g, 1.25 mmol), 1N NaOH (1.4 ml, 1.4 mmol) and MeOH (5ml) to give the product (0.76 g).

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-phenylcarbamoyl-L-lysyl-L-valyl-L-prolyl-L-valinol

A solution of the product of Example 4b (0.76 g, 1.3 mmol) in dry THF(10 ml) was cooled to -20° C. under a nitrogen atmosphere.N-methylmorpholine (0.15 ml, 1.3 mmol) was added followed by isobutylchloroformate (0.17 ml, 1.3 mmol). The reaction mixture was stirred for10 min. and the temperature lowered to -45° C. A solution of L-valinol(0.135 g, 1.3 mmol) in dry THF (5 ml) was added. The reaction mixturewas allowed to warm to room temperature and stirred overnight. Thereaction mixture was filtered, concentrated under vacuum, and theresidue purified by flash chromatography on silica gel with MeOH:EtOAc(10:90) to give the product (0.68 g); TLC, R_(f) =0.41, silica gel,MeOH:EtOAc (1:9).

d.N-alpha-t-Butyloxycarbonyl-N-epsilon-phenylcarbamoyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The title compound was prepared as in Example 1f, using oxalyl chloride(0.12 ml, 1.78 mmol) in CH₂ Cl₂ (0.5 ml) and adding DMSO (0.32 ml, 3.56mmol) in CH₂ Cl₂ (0.5 ml), the product of Example 4c (0.6 g, 0.89 mmol)in CH₂ Cl₂ (1 ml) and TEA (0.49 ml, 3.56 mmol). The crude product waspurified by flash chromatography on silica gel with MeOH:Et₂ O (10:90)to give the product (0.41 g) as a white foam; TLC, R_(f) =0.43, silicagel, MeOH:Et₂ O (1:9).

EXAMPLE 5N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOOCH₂--C₆ H₅ ; R⁴ ═--CH₂ CH₂ COOH.

a.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinol

A solution of the product of Example 1e (3.57 g, 5.4 mmol) andtrifluoroacetic acid (4.2 ml, 54 mmol) in CH₂ Cl₂ (10 ml) was stirred atroom temperature for 2 hr. The reaction mixture was concentrated undervacuum, the residue was dissolved in CH₃ CN (90 ml), andN-methylmorpholine (6.0 ml, 54 mmol) was added. The reaction mixture wascooled to 0° C. and a solution of succinic anhydride (0.59 g, 5.9 mmol)in CH₃ CN (10 ml) was added dropwise. The reaction mixture was kept at5° C. for 72 hr. and then concentrated under vacuum. The residue wasdissolved in EtOAc, washed successively with aqueous 1N HCl and brine,dried over Na₂ SO₄ and filtered. The EtOAc solution was concentratedunder vacuum to give the product as a viscous yellow oil; TLC, R_(f)=0.4, silica gel, MeOH:CHCl₃ (1:9). The yellow oil obtained, will, onstanding, slowly convert to an unidentified secondary product (TLC,R_(f) =0.6, silica gel, MeOH:CHCl₃ (1:9)) which can be re-converted tothe desired product by treatment with aqueous MeOH containing 1.1equivalents of NaOH.

b.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The title compound was prepared as described in Example 1f using oxalylchloride (0.175 ml, 2.0 mmol) in dry CH₂ Cl₂ (1 ml) and adding DMSO(0.28 ml, 4.0 mmol) in CH₂ Cl₂ (0.5 ml), the product of Example 5a (0.7g, 1.06 mmol) in CH₂ Cl₂ (2ml) and TEA (0.55 ml, 4.0 mmol) to give thecrude product (0.660 g) as a pale yellow foam. Purification by flashchromatography on silica gel (Baker, acidic) using an eluent ofMeOH:CHCl₃ (5:95) gave the product (0.188 g); TLC, R_(f) =0.45, silicagel, MeOH:CHCl₃ :HOAc (9.5:90:0.5).

Elemental Analysis: Calculated for C₃₃ H₄₉ N₅ O₉.1/2H₂ O: C, 59.26; H,7.53; N, 10.47. Found: C, 59.48; H, 7.27; N, 10.31.

¹ H-NMR (dimethyl sulfoxide-d₆): delta 9.45 (1H, s), 8.16 (1H, d), 7.98(1H, d), 7.82 (1H, d), 7.34 (5H, m), 7.22 (1H, dd), 5.0 (2H, s), 4.44(1H, m), 4.26 (2H, m), 4.07 (1H, m), 3.74 (1H, m), 3.56 (1H, m), 2.96(2H, m), 2.4 (4H, m), 2.26-1.16 (12H, m), 0.86 (12H, m).

EXAMPLE 6N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal--AlternateSynthesis

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOOH₂ --C₆H₅ ; R⁴ ═--(CH₂)₂ CO₂ H

a. N-Benzyloxycarbonyl-L-valyl-L-proline t-butyl ester

A solution of N-benzyloxycarbonyl-L-valine (56.25 g, 0.0244 mol) andHOBT (60.67 g, 0.45 mol) in DMF (565 ml) was cooled to 5° C. DCC (50.89g, 0.247 mol) was added in one portion. The mixture was stirred anadditional 15 min. at 5° C. and then L-proline t-butyl ester (38.36 g,0.224 mol) was added. The mixture was stirred an additional 2 hr. at 5°C. then for 48 hr. at room temperature. The mixture was filtered andconcentrated under vacuum. The oily residue was dissolved in EtOAc (1liter) and washed successively with 20% aqueous citric acid, saturatedaqueous NaHCO₃ and brine. The organic phase was dried over Na₂ SO₄,filtered and concentrated under vacuum to afford the product (92 g) as awhite foam; TLC, R_(f) =0.9 silica gel, CHCl₃ /EtOAc (85:15).

b. L-Valyl-L-proline t-butyl ester

A mixture of the product of Example 6a (92 g. 0.227 mol) and 10% Pd/C(10 g) in EtOH (1 liter) was hydrogenated on a Parr shaker for 6 hr. at60 psi at room temperature. The mixture was filtered through Celite® andconcentrated under vacuum to afford the product (62 g) as a viscousyellow oil; TLC, R_(f) =0.3, silica gel, MeOH/CHCl₃ (10:90).

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolinet-butyl ester

HOBT (8.55 g, 0.06 mol) and DCC (7.35 g, 0.036 mol) were added to asolution ofN-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysine (12.0 g,0.03 mol) in dry THF (200 ml) at 5° C. The mixture was stirred for 15min. at 5° C. To the mixture was then added the product of Example 6b(9.16 g, 0.03 mol). The resulting solution was allowed to warm slowly toroom temperature and was stirred for 24 hr. The mixture was filtered andconcentrated under vacuum. The oily residue was dissolved in EtOAc (500ml) and washed successively with 20% aqueous citric acid, saturatedaqueous NaHCO₃ and brine. The organic phase was dried over Na₂ SO₄,filtered and concentrated under vacuum to give the product (20 g) as ayellow foam; TLC, R_(f) =0.6, silica gel, CHCl₃ :MeOH (95:5).

d. N-epsilon-Benzyloxycarbonyl-L-lysyl-L-valyl-L-prolinetrifluoroacetate salt

Trifluoroacetic acid (25 ml, 0.32 mol) was added to a solution of theproduct of Example 6c (10.38 g, 16.4 mmol) in CH₂ Cl₂ (25 ml) at roomtemperature and the resulting mixture was stirred for 5 hr. The solutionwas concentrated under vacuum and the residue triturated with Et₂ O. Theproduct was isolated by filtration and dried under vacuum to give awhite solid (7.4 g).

e.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxy-carbonyl-L-lysyl-L-valyl-L-proline

Aqueous 1N NaOH (460 ml, 0.46 mol) was added dropwise to a solution ofthe product of Example 6d (136.5 g, 0.23 mol) in CH₂ Cl₂ (1.5 liters) atroom temperature. The mixture was cooled to 0° C. and3-carbomethoxypropionyl chloride (34.6 g, 0.23 mol) was added dropwise.The resulting solution was stirred vigorously for 12 min. The solutionwas removed from the ice bath, diluted with H₂ O (800 ml), and adjustedto pH 2.0 by dropwise addition of aqueous 1N HCl (40 ml). The organicphase was separated and the aqueous layer was extracted with CH₂ Cl₂(2×500 ml). The organic layers were combined, washed successively withH₂ O (2×500 ml) and brine (500 ml), dried over Na₂ SO₄ and filtered. Thefiltrate was concentrated under vacuum to give the crude product (126.4g); TLC, R_(f) =0.25, silica gel, MeOH/CHCl.sub. 3 /HOAC (5:94.5:0.5).

f. N-Benzyloxycarbonyl-L-valinol

Benzyl chloroformate (91.0 g, 0.532 mol, 95% purity) was added dropwiseover a period of 1 hr. to a pre-cooled (0° C.) solution of L-valinol(50.0 g, 0.484 mol) and triethylamine (60.0 g, 0.6 mol) in CHCl₃ (1500ml). The reaction mixture was stirred for 1 hr. at 0° C. and thenallowed to warm to room temperature over 2 hr. The reaction mixture wasconcentrated under vacuum. EtOAc (1500 ml) was added to the resultingresidue and the organic solution was washed with aqueous 1N NaOH andbrine. The organic phase was dried over MgSO₄, filtered and concentratedunder vacuum. The resulting residue was purified by flash chromatographyon a column of silica gel (6 cm×30 cm) using a stepwise gradient of Et₂O:hexane (1:5) followed by pure Et₂ O to give the product (91.4 g) as awhite waxy solid; TLC, R_(f) =0.23, silica gel, hexane:Et₂ O (50:50).

g. N-Benzyloxycarbonyl-L-valinal

A solution of DMSO (107.2 g, 1.372 mol) in CH₂ Cl₂ (150 ml) was addeddropwise over 0.5 hr. to a pre-cooled (-60° C.), stirred solution ofoxalyl chloride (87.1 g, 0.686 mol) in CH₂ Cl₂ (800 mls) under anitrogen atmosphere. The temperature of the mixture rose to -45° C. Thereaction mixture was then warmed to -30° C. A solution of the product ofExample 6f (81.5 g, 0.343 mol) in CH₂ Cl₂ (300 ml) was added dropwiseover 45 min. at -30° C. The reaction mixture was stirred for 50 min. at-25° C., cooled to -40° C. and a solution of diisopropylethyl amine(177.4 g, 1.372 mol) in CH₂ Cl₂ (250 ml) was added dropwise over 45 min.at -40° C. The reaction mixture was stirred for 1 hr. as it warmed toroom temperature. The reaction mixture was diluted with CH₂ Cl₂ (1500ml) and the organic phase was washed with aqueous 1N HCl and thenconcentrated under vacuum to give the product (98 g) as a green oilwhich was used immediately without further purification; TLC, R_(f)=0.48, silica gel, hexane:Et₂ O (50:50).

h. N-Benzyloxycarbonyl-L-valinal diethylacetal

Triethyl orthoformate (700 g, 4.723 mol), absolute EtOH (800 ml) andp-toluenesulfonic acid monohydrate (5.0 g, 0.026 mol) were added to theproduct of Example 6g (81 g, 0.343 mol). The mixture was stirred for 10minutes at room temperature and then concentrated under vacuum. Theresulting residue was dissolved in Et₂ O and washed with saturatedaqueous NaHCO₃. The organic phase was dried over Na₂ SO₄, filtered andconcentrated under vacuum to give the crude product. This product waspurified by flash chromatography with silica gel using a stepwisegradient of hexane through mixtures of CH₂ Cl₂ :hexane to EtOAc:CH₂ Cl₂(30:70) to give the product as a pale yellow oil; TLC, R_(f) =0.21,silica gel, CH₂ Cl₂ :petroleum ether (50:50).

i. L-Valinal diethylacetal

A mixture of the product of Example 6h (147.8 g, 0.478 mol) and 10% Pd/C(10 g) in EtOAc (1500 ml) was stirred under H₂ (1 atm.) until 2500 ml ofH₂ was consumed. Twice during this time the reaction was interrupted and10% Pd/C (10 g) was added. The reaction mixture was then filteredthrough a pad of Celite®. 10% Pd/C (10 g) was added and the reactionmixture stirred until 10.92 liters of H₂ was consumed. The reactionmixture was filtered through Celite® and the filtrate was concentratedunder vacuum to give the product (78.8 g) as a pale yellow oil;[alpha]_(D) ²⁵ +7.8.

j.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Isobutyl chloroformate (27.32 g, 0.20 mol) was added to a solution ofthe product of Example 6e (118 g, 0.20 mol) and N-methylmorpholine(20.23 g, 0.20 mol) in dry THF (1500 ml) at -15° C. The reaction mixturewas stirred for 0.5 hr. at -15° C. and then cooled to -40° C. A solutionof the product of Example 6i (35.05 g, 0.20 mol) in dry THF (250 ml) wasadded dropwise. The resulting mixture was allowed to warm slowly to roomtemperature and stirred overnight. The reaction mixture was filtered andthe filtrate was concentrated under vacuum. The residue was partiallypurified by flash chromatography on silica gel with a stepwise gradientfrom pure Et₂ O to MeOH:Et₂ O (10:90). Final purification on a WatersPrep 500® Liquid Chromatograph equipped with silica gel columns usingMeOH:Et₂ O:CH₂ Cl₂ (2.5:40:60) as eluent gave the product (87.5 g, 58%).

k.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A solution of aqueous 1N NaOH (14 ml, 14 mmol) was added to a solutionof the product of Example 6j (10.0 g, 13.4 mmol) in MeOH/H₂ O (1:1, 280ml) at room temperature. The mixture was stirred for 2-4 hrs. The MeOHwas removed under vacuum to give an aqueous solution.

Dowex® 50WX8-H resin (500 g), H₂ O (1000 ml) and acetone (120 ml) wereadded to this aqueous solution. This mixture was stirred at roomtemperature for 24 hr. The mixture was filtered and the resin was washedwith acetone (1.0 liter). The filtrate and the washings were combinedand the acetone was removed under vacuum. The H₂ O was removed using alyophilizer to give the product (7.8 g, 11.8 mmol) as a white, fluffysolid; TLC, R_(f) =0.3, silica gel, MeOH:CHCl₃ :HOAc (5:95:0.5).

1. Sodium salt ofN-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A solution of the product of Example 6k (100 mg, 0.150 mmol) in water(100 ml) was treated with aqueous 1N NaOH (0.15 ml) and then stirred for10 min. at room temperature. The water was removed with a lyophilizer togive the sodium salt as a white powder.

¹ H-NMR (dimethyl sulfoxide-d₆): delta 9.43 (1H, s), 8.57 (1H, d, J=7.5Hz), 8.20 (1H, d, J=7.6 Hz), 8.03 (1H, d, J=7.6 Hz), 7.34 (6H, m), 4.99(2H, s), 4.44 (1H, m), 4.29 (1H, m), 4.13 (1H, m), 4.02 (1H, m), 3.68(1H, m), 3.51 (1H, m), 2.95 (2H, m), 2.36-1.16 (16H, m), 0.86 (12H, m).

Other acids of the invention may also be converted to theircorresponding sodium salts by this procedure.

m. Dilysine adduct ofN-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

L-Lysine (113 mg, 0.778 mmol) was added to a solution of the product ofExample 6k (250 mg, 0.389 mmol) in MeOH:H₂ O (1:1, 10 ml). The mixturewas stirred at room temperature for 10 min. The methanol was removedunder vacuum and the aqueous solution was lyophylized to give theproduct (370 mg) as a white solid.

Elemental Analysis. Calculated for C₄₅ H₇₆ N₉ O₁₃.2H₂ O: C, 54.75; H,8.16; N, 12.77. Found: C, 54.64; H, 8.08; N, 12.58.

Adducts of other compounds of the invention may be prepared in a similarmanner. If a carboxyic acid group is not present in the molecule, onlyone equivalent of the amine is used.

This Example 6 illustrates the preferred method for preparing the titlecompound.

EXAMPLE 7N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-D-valinal

Formula I: R¹ ═(D)--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NHCOOCH₂C₆ H₅ ; R⁴ ═--(CH₂)₂ COOH

a.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-D-valinaldiethylacetal

HOBT (1.85 g, 13.68 mmol) was added to a solution of the product ofExample 6e (4.04 g, 6.84 mmol) and D-valinal diethylacetal (1.2 g, 6.85mmol) in dry THF (70 ml) at 0° C. under a nitrogen atmosphere and themixture was stirred for 15 min. To the resulting solution was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.44 g,7.52 mmol) followed by N-methylmorpholine (1.03 g, 10.26 mmol). Themixture was stirred for one hr. at 0° C. and then for 48 hr. at roomtemperature. The reaction mixture was concentrated in vacuo and theresulting residue was distributed between EtOAc and H₂ O. The organiclayer was isolated and washed successively with aqueous 1N HCl, brine,saturated aqueous NaHCO₃ and brine, dried over MgSO₄, filtered andconcentrated to give a yellow oil. This oil was purified by flashchromatography on silica gel with CH₂ Cl₂ :Et₂ O:MeOH (60:40:3) to givea white foam (2.3 g); TLC, R_(f) =0.4 silica gel, CH₂ Cl₂ :Et₂ O:MeOH(60:40:3).

b.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-D-valinal

1N NaOH (3.31 ml) was added to a solution of the product of Example 7a(2.3 g, 3.01 mmol) in MeOH:H₂ O (1:1, 40 ml) at room temperature under anitrogen atmosphere and the mixture was stirred for 2.5 hr. The MeOH wasremoved in vacuo and the resulting aqueous solution was diluted withadditional H₂ O (160 ml) and acetone (20 ml), treated with Dowex® 50WX8-H resin (120 ml) and stirred for 24 hr. The mixture was filtered andthe resin was washed with acetone. The combined filtrates wereconcentrated to remove the acetone and the H₂ O was lyophilized to givea fluffy white solid (1.3 g); TLC, R_(f) =0.7, reverse-phase C₁₈ (i.e.,octadecylsilane), MeOH:H₂ O (75:25).

Elemental Analysis. Calculated for C₃₃ H₄₉ N₅ O₉.0.25H₂ O: C, 59.66; H,7.51; N, 10.54. Found: C, 59.51; H, 7.24; N, 10.54.

EXAMPLE 8N-alpha-Methoxysuccinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NH--COOCH₂C₆ H₅ ; R⁴ ═--(CH₂)₂ --COOCH₃

a.N-alpha-Methoxysuccinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinol

A solution of the product of Example 1e (3.2 g, 4.84 mmol) andtrifluoroacetic acid (3.7 ml, 48 mmol) in CH₂ Cl₂ (4 ml) was stirred atroom temperature for 24 hr. The reaction mixture was concentrated undervacuum, the residue dissolved in THF (60 ml) and cooled to 0° C. underan atmosphere of dry N₂. N-methylmorpholine (8.2 ml, 75 mmol) was added,then a solution of 3-carbomethoxypropionyl chloride (0.620 ml, 5.0 mmol)in 10 ml THF was added dropwise. The reaction mixture was stirred at 0°C. for 1 hr., then filtered and concentrated under vacuum. The residuewas dissolved in EtOAc, washed successively with H₂ O, aqueous 1N HCl,saturated aqueous NaHCO₃ and brine, dried over anhydrous Na₂ SO₄ andfiltered. The filtrate was concentrated under vacuum to give 3.1 g of anoff-white foam. Purification by flash chromatography on silica gel withMeOH:EtOAc (5:95-10:90) gave the desired product (0.95 g); TLC, R_(f)=0.35, silica gel, MeOH:EtOAc (1:9).

b.N-alpha-Methoxysuccinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The title compound was prepared as described in Example 1f using oxalylchloride (0.22 ml, 2.54 mmol) in dry CH₂ Cl₂ (5 ml) and adding DMSO(0.36 ml, 5.08 mmol) in dry CH₂ Cl₂ (2 ml), the product of Example 8a(0.86 g, 1.27 mmol) in dry CH₂ Cl₂ (3 ml) and TEA (0.7 ml, 5.08 mmol) togive the crude product as a pale yellow foam. Purification by flashchromatography on silica gel with MeOH:EtOAc (5:95) gave the product(0.73 g); TLC, R_(f) =0.58, silica gel, MeOH:CHCl₃ (1:9).

EXAMPLE 9N-alpha-(4-Carboxybutyryl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NH--CO₂ CH₂C₆ H₅ ; R⁴ ═--(CH₂)₃ CO₂ H.

a.N-alpha-(4-Carbomethoxybutyryl)-N-epsilon-benzyloxy-carbonyl-L-lysyl-L-valyl-L-proline

Aqueous 1N NaOH (11.86 ml) was added dropwise to a stirred solution ofthe product of Example 6d (3.5 g, 5.93 mmol) in CH₂ Cl₂ (70 ml) at roomtemperature. The mixture was cooled to 0° C. and 4-carbomethoxybutyrylchloride (0.82 ml, 5.93 mmol) was added dropwise. The resulting mixturewas stirred vigorously for 12 min. The solution was diluted with H₂ O(30 ml), and adjusted to pH 2.0 by dropwise addition of aqueous 1N HCl.The organic layer was collected and the aqueous layer was extractedtwice with CH₂ Cl₂. The combined organic layers were washed successivelywith H₂ O and brine, dried over Na₂ SO₄, filtered and concentrated undervacuum to give the product (3.23 g); TLC, R_(f) =0.25, silica gel,MeOH:CHCl₃ :HOAC (0.45:9.5:0.1).

b.N-alpha-(4-Carbomethoxybutyryl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Isobutyl chloroformate (0.702 ml, 5.42 mmol) was added to a solution ofthe product of Example 9a (3.2 g, 5.42 mmol) and N-methylmorpholine(0.595 ml, 5.42 mmol) in dry THF (50 ml) at -15° C. The reaction mixturewas stirred for 0.5 hr. at -15° C. and then cooled to -40° C. A solutionof L-valinal diethylacetal (0.948 g, 5.42 mmol) in dry THF (7 ml) wasadded dropwise. The resulting mixture was allowed to warm slowly to roomtemperature and was stirred overnight. The solution was filtered, thefiltrate was concentrated under vacuum, and the product was purified byflash chromatography on silica gel with Et₂ O:CH₂ Cl₂ :MeOH (4:6:3) togive a foam (1.5 g).

Elemental Analysis. Calculated for C₃₉ H₆₃ N₅ O₁₀.0.5H₂ O: C, 60.76; H,8.36; N, 9.08. Found: C, 60.92; H, 8.25; N, 9.04.

c.N-alpha-(4-Carboxybutyryl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A mixture of the product of Example 9b (0.75 g, 0.985 mmol) in CH₃ OH:H₂O (1:1, 20 ml) and aqueous 1N NaOH (1.1 ml) was stirred at roomtemperature for 4 hr. The methanol was removed under vacuum to give anaqueous solution of the product. Dowex® 50WX8-H resin (38 ml), H₂ O (100ml), acetone (25 ml) were added and the resulting mixture was stirred atroom temperature for 48 hr. The mixture was filtered and the resin waswashed with acetone (100 ml). The filtrates were combined andconcentrated under vacuum to give an aqueous solution which waslyophilized to give the crude product. The product was purified on areverse-phase column (C₁₈) with MeOH:H₂ O (60:40) to give a white solid(0.4 g); TLC, R_(f) =0.5, silica gel, MeOH:CHCl₃ :HOAc (0.45, 9.5, 0.1).

EXAMPLE 10N-alpha-N-epsilon-Bis-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NHCOOCH₂C₆ H₅ ; R⁴ ═--OCH₂ C₆ H₅.

a. N-alpha-N-epsilon-Bis-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolinet-butyl ester

HOBT (5.0 g, 0.037 mol) was added to a solution ofN-alpha-N-epsilon-bis-benzyloxycarbonyl-L-lysine (7.66 g, 0.0185 mol)and the product of Example 6b (5.0 g, 0.0185 mol) in 75 ml dry THF at 0°C. under a nitrogen atmosphere. This mixture was stirred for 15 min. andDCC (4.19 g, 0.0203 mol) was then added. The mixture was stirred for 30min. at 0° C. and then at room temperature for 3 days. The reactionmixture was filtered and the filtrate was concentrated under vacuum. Theresulting residue was partitioned between EtOAc (200 ml) and 20% aqueouscitric acid (75 ml). The organic layer was isolated and washed with 20%aqueous citric acid (2×75 ml), brine, saturated aqueous NaHCO₃ andbrine, dried over MgSO₄, filtered and concentrated. The residue waspurified by flash chromatography on silica gel with CH₂ Cl₂ :THF:HOAc(90:10:1) to give a white foam (8.93 g); TLC, R_(f) =0.60 silica gel,CH₂ Cl₂ :MeOH (95:5).

b. N-alpha-N-epsilon-Bis-benzyloxycarbonyl-L-lysyl-L-valyl-L-proline

Trifluoroacetic acid (15 ml) was added to a solution of the product ofExample 10a (8.93 g) in CH₂ Cl₂ (50 ml) at room temperature under anitrogen atmosphere. The reaction mixture was stirred for 4 hr., thendiluted with toluene (200 ml) and concentrated in vacuo to give a foam(5.73 g).

c.N-alpha-N-epsilon-Bis-benzyloxycarbonyl-L-lysl-L-valyl-L-prolyl-L-valinaldiethylacetal

HOBT (1.29 g, 9.56 mmol) was added to a solution of the product ofExample 10b (3.0 g, 4.78 mmol) and L-valinal diethylacetal (0.84 g, 4.78mmol) in dry THF (65 ml) at 0° C. under a nitrogen atmosphere. Afterthis mixture was stirred for 15 min,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.0 g, 5.26mmol) and N-methylmorpholine (0.72 g, 7.17 mmol) were added. The mixturewas stirred for one hour at 0° C. and then at room temperature for 20hr. The reaction mixture was concentrated in vacuo and the resultingresidue was partitioned between EtOAc (100 ml) and H₂ O (50 ml). Theisolated organic layer was washed with 20% aqueous citric acid, brine,saturated aqueous NaHCO₃ and brine, then dried over MgSO₄, filtered andconcentrated. The product was purified by flash chromatography on silicagel with CH₂ Cl₂ :Et₂ O:MeOH (60:40:2) to give a white foam (2.2 g); TLCR_(f) =0.45, silica gel, CH₂ Cl₂ :Et₂ O:MeOH (60:40:2).

d.N-alpha-N-epsilon-Bis-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A mixture of p-toluenesulfonic acid (0.07 g, 0.37 mmol) and the productof Example 10c in acetone (100 ml) was stirred for 3 hr. at roomtemperature under a nitrogen atmosphere. The mixture was concentrated invacuo and the residue was dissolved in EtOAc (50 ml). This solution waswashed with 5% aqueous NaHCO₃ and brine, dried over MgSO₄, filtered andconcentrated to give a white foam (0.82 g); TLC, R_(f) =0.46 silica gel,CH₂ Cl₂ :MeOH (95:5).

Elemental Analysis. Calculated for C₃₇ H₅₁ N₅ O₈.0.4H₂ O: C, 63.39; H,7.44; N, 9.89. Found: C, 63.43; H, 7.43; N, 9.77.

EXAMPLE 11 N-Succinyl-L-leucyl-l-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--CH₂ CH(CH₃)₂ ; R⁴═--(CH₂)₂ COOH

a. N-t-Butyloxycarbonyl-L-leucyl-L-valyl-L-proline t-butyl ester

HOBT (3.10 g, 0.0229 mol) was added to a solution ofN-t-butyloxycarbonyl-L-leucine (2.65 g, 0.0115 mol) in dry THF (75 ml)at 0° C. under a nitrogen atmosphere and the reaction mixture wasstirred for 15 min. To the resulting solution was added a suspension ofthe product of Example 6b (5.10 g, 0.0115 mol) in dry THF followed byDCC (2.60 g, 0.0126 mol). The mixture was stirred for 1 hr. at 0° C. andthen at room temperature for 4 days. The reaction mixture was filteredand the filtrate concentrated under vacuum. The resulting residue wasmixed with cold CHCl₃ (500 ml) and filtered. The filtrate was washedsuccessively with 5% aqueous NaHCO₃, 20% aqueous citric acid, and brine,dried over Na₂ SO₄, filtered and concentrated to give 3.72 g of thecrude product. This material was purified by flash chromatography onsilica gel with CHCl₃ give the product (2.19 g) as a white waxy solid;TLC, R_(f) =0.50, silica gel, MeOH:CHCl₃ (5:95).

b. L-Leucyl-L-valyl-L-proline trifluoroacetic acid salt

Trifluoroacetic acid (18.59 g, 22 ml, 0.163 mol) was added to a solutionof the product of Example 11a (3.94 g, 8.2 mol) in dry CH₂ Cl₂ (22 ml)at room temperature under a nitrogen atmosphere. The reaction mixturewas stirred for 5 hr. The resulting solution was concentrated undervacuum and the residual amber oil was triturated with Et₂ O to affordthe product (3.07 g) as a white solid.

c. N-(3-Carbomethoxypropionyl)-L-leucyl-L-valyl-L-proline

A solution of the product of Example 11b (3.06 g, 6.9 mmol) in dry CH₂Cl₂ (60 ml) at room temperature under a nitrogen atmosphere was treatedwith aqueous 1N NaOH (15 ml). The solution was cooled to 0° C.,3-carbomethoxypropionyl chloride (1.04 g, 6.9 mmol) was added and thereaction mixture was stirred vigorously for 15 min. The reaction mixturewas diluted with H₂ O (50 ml) and adjusted to pH 2 with aqueous 1N HCl.The organic phase was separated and the aqueous phase was extracted withCH₂ Cl₂. The combined organic phases were dried over Na₂ SO₄, filteredand concentrated under vacuum to give the crude product (2.58 g). Thismaterial was purified by flash chromatography on silica gel withMeOH:CHCl₃ (5:95) to give the product (1.30 g) as a white foam; TLC,R_(f) =0.35, silica gel, MeOH:CHCl₃ :HOAc (5:94.5:0.5).

d. N-(3-Carbomethoxypropionyl)-L-leucyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Isobutyl chloroformate (0.38 g, 2.8 mmol) was added to a solution of theproduct of Example 11c (1.16 g, 2.6 mmol) and N-methylmorpholine (0.40g, 3.9 mmol) in dry THF (75 ml) at -10° C. under a nitrogen atmosphere.A white precipitate formed immediately. The reaction mixture was stirredfor 20 min and then cooled to -40° C. L-Valinal diethylacetal (0.46 g,2.6 mmol) in dry THF (8 ml) was added in one portion. The reactionmixture was warmed slowly to room temperature and stirred overnight. Theresulting mixture was filtered and the filtrate concentrated undervacuum to give a pale yellow oil (1.77 g). The crude material waspurified by flash chromatography on silica gel with CH₂ Cl₂ :Et₂ O:MeOH(60:37:3) as eluent to give the product (1.03 g) as a white foam; TLC,R_(f) =0.52, silica gel, MeOH:CHCl₃ (5:95).

e. N-Succinyl-L-leucyl-L-valyl-L-prolyl-L-valinal diethylacetal sodiumsalt

A stirred solution of the product of Example 11d (0.98 g, 1.6 mmol) inMeOH:H₂ O (1:1, 30 ml) at room temperature under a nitrogen atmospherewas treated with aqueous 1N NaOH (1.72 ml). The reaction mixture wasstirred for 2 hr. and then concentrated under vacuum to give an aqueoussolution of the product; TLC, R_(f) =0.14, silica gel, MeOH:CHCl₃(5:95).

f. N-Succinyl-L-leucyl-L-valyl-L-prolyl-L-valinal

Dowex® 50WX8-H resin (50 ml) was added to a stirred solution of theproduct of Example 11e (1.6 mmol) in H₂ O (100 ml) at room temperatureunder a nitrogen atmosphere. The reaction mixture was stirred for 2days. The reaction mixture was filtered and the resin was washed withacetone (50 ml). The filtrate and washes were concentrated under vacuumto remove the acetone. The aqueous mixture was lyophilized to give theproduct (0.78 g) as a white powder; TLC, R_(f) =0.24, silica gel,MeOH:CHCl₃ :HOAc (5:94.5:0.5).

EXAMPLE 12N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-leucinal

Formula (I): R¹ ═--CH₂ CH (CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄NHCOO--CH₂ C₆ H₅ ; R⁴ ═--OC(CH₃)₃

a.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-leucinol

The title compound was prepared as in Example 1e except that a solutionof the product of Example 1d (2.2 g, 3.8 mmol) and N-methylmorpholine(0.39 g, 3.8 mmol) in dry THF (20 ml) was used and isobutylchloroformate (0.525 g, 3.8 mmol) was added followed by a solution ofL-leucinol (0.44 g, 3.8 mmol) in dry THF (20 ml). The crude product waspurified by flash chromatography on silica gel with MeOH/Et₂ O(2.5:97.5) followed by MeOH/Et₂ O (5:95) to give the product (2.16 g);TLC, R_(f) =0.29, silica gel, MeOH:Et₂ O (5:95).

b.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-leucinal

The title compound was prepared as in Example 1f, except that oxalylchloride (0.614 g, 4.8 mmol) in CH₂ Cl₂ (8.0 ml) was used. DMSO (0.75 g,9.6 mmol) in CH₂ Cl₂ (4.0 ml), a solution of the product of Example 12a(1.63 g, 2.4 mmol) in CH₂ Cl₂ (4.0 ml) and TEA (1.29 g, 9.6 mmol) in CH₂Cl₂ (4.0 ml) were added successively to give the crude product, whichwas purified by flash chromatography on silica gel with MeOH:CHCl₃(2.5:97.5) to give the product (1.44 g); TLC, R_(f) =0.53, silica gel,MeOH:CHCl₃ (10:90).

EXAMPLE 13N-(3-Carbomethoxypropionyl)-L-norleucyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; Rhu 2═--CH(CH₃)₂ ; R³ ═--(CH₂)₃ CH₃ ; R⁴═--(CH₂)₂ COOCH₃

a. N-Benzyloxycarbonyl-L-valyl-L-proline

Trifluoracetic acid (70 ml, 0.90 mol) was added to a solution of theproduct of Example 6a (16.5 g, 39.2 mmol) in CH₂ Cl₂ (100 ml) at roomtemperature and the resulting mixture was stirred for 3 hr. The solutionwas diluted with toluene (100 ml) and concentrated under vacuum. Theresidue was taken up in toluene and reconcentrated 5 times to finallygive the product (12.85 g) as a tan solid; TLC, R_(f) =0.45, silica gel,MeOH:CH₂ Cl₂ (5:95).

b. N-Benzyloxycarbonyl-L-valyl-L-prolyl-L-valinal diethylacetal

HOBT (4.21 g, 31.1 mmol) was added to a solution of the product ofExample 13a (5.17 g, 15.55 mmol) and L-valinal diethylacetal (2.73 g,15.55 mmol) in dry THF (75 ml) at 0° C. under a nitrogen atmosphere.This solution was stirred for 15 min and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (3.28 g,17.1 mmol) followed by N-methylmorpholine (2.36 g, 23.3 mmol) wereadded. The stirred for 1 hr. at 0° C. and for 3 days at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was partitioned between EtOAc and H₂ O. The organic layer wasisolated and washed with aqueous 1N HCl, brine, saturated aqueous NaHCO₃and brine, then dried over MgSO₄, filtered and concentrated to give aviscous oil. The product was purified by flash chromatography on silicagel with CH₂ Cl₂ :Et₂ O:MeOH (75:25:0.5) to give a colorless oil (4.4g); TLC, R_(f) =0.55, silica gel, CH₂ Cl₂ :Et₂ O:MeOH (75:25:1).

c. L-Valyl-L-prolyl-L-valinal diethylacetal

The product of Example 13b (3.63 g, 7.18 mmol) and 10% Pd/C (0.5 g) inEtOH (75 ml) was hydrogenated on a Parr shaker (3 atm H₂). When thetheoretical amount of H₂ was consumed the mixture was filtered throughCelite® and concentrated under vacuum to give the product (2.5 g); TLC,R_(f) =0.3, silica gel, MeOH:CH₂ Cl₂ (1:9).

d. N-Benzyloxycarbonyl-L-norleucyl-L-valyl-L-prolyl-L-valinaldiethylacetal

HOBT (2.48 g, 18.3 mmol) was added to a solution of the product ofExample 13c (3.4 g, 9.15 mmol) and N-benzyloxycarbonyl-L-norleucine(2.43 g, 9.15 mmol) in dry THF (75 ml) at 0° C. under a nitrogenatmosphere. This mixture was stirred for 15 min and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.93 g,10.06 mmol) followed by N-methyl-morpholine (1.39 g, 13.73 mmol) wereadded. The mixture was stirred for 1 hr. at 0° C. and then 24 hr. atroom temperature. The reaction mixture was concentrated in vacuo and theresulting residue was partitioned between EtOAc and H₂ O. The isolatedorganic layer was washed with aqueous 1N HCl, brine, saturated aqueousNaHCO₃ and brine, then dried over MgSO₄, filtered and concentrated togive the crude product. Purification by flash chromatography on silicagel with CH₂ Cl₂ :MeOH (95:5) gave the product as a white foam (4.76 g);TLC, R_(f) =0.65 silica gel, CH₂ Cl₂ :MeOH (9:1).

Elemental Analysis. Calculated for C₃₃ H₅₄ N₄ O₇ : C, 64.05; H, 8.79; N,9.05. Found: C, 63.74; H, 8.78; N, 9.00.

e. L-Norleucyl-L-valyl-L-prolyl-L-valinal diethylacetal

The product of Example 13d (3.70 g, 5.98 mmol) and 10% Pd on carbon(0.25 g) in EtOH (50 ml) was hydrogenated on a Parr shaker (3 atm H₂).After the theoretical amount of H₂ was consumed the mixture was filteredthrough Celite® and concentrated under vacuum to give the product (3.2g); TLC, R_(f) =0.25, silica gel, CH₂ Cl₂ :MeOH (95:5).

f. N-(3-Carbomethoxypropionyl)-L-norleucyl-L-valyl-L-prolyl-L-valinaldiethylacetal

A mixture of aqueous 1N NaOH (6.2 ml) and the product of Example 13e(3.0 g, 6.19 mmol) in CH₂ Cl₂ (30 ml) was stirred vigorously at 0° C.under a nitrogen atmosphere. After addition of 3-carbomethoxypropionylchloride (0.93 g, 6.19 mmol) in one batch, the reaction mixture wasstirred for 15 min. then diluted with H₂ O (25 ml) and adjusted to pH 2with aqueous 1N HCl. The mixture was extracted with EtOAc. The combinedextracts were washed with brine, dried over MgSO₄, filtered andconcentrated to give a foam. The product was purified by flashchromatography on silica gel with CH₂ Cl₂ :Et₂ O:MeOH (60:40:2) to givea white foam (2.41 g); TLC, R_(f) =0.45 silica gel, CH₂ Cl₂ :Et₂ O:MeOH(60:40:3).

Elemental Analysis. Calculated for C₃₀ H₅₄ N₄ O₈ : C, 60.18; H, 9.09; N,9.36. Found: C, 60.00; H, 8.91; N, 9.22.

g. N-(3-Carbomethoxypropionyl)-L-norleucyl-L-valyl-L-prolyl-L-valinal.

A mixture of the product of Example 13f (0.50 g, 0.835 mmol) andp-toluenesulfonic acid (0.039 g, 0.208 mmol) in acetone (50 ml) wasstirred for 3 hr. under a nitrogen atmosphere. The mixture wasconcentrated in vacuo and the resulting residue was extracted intoEtOAc. This solution was washed successively with 5% aqueous NaHCO₃ andbrine, dried over MgSO₄, filtered and concentrated to give a foam (0.39g); TLC, R_(f) =0.5, silica gel, CH₂ Cl₂ :EtOAc (1:1).

EXAMPLE 14N-Acetylglycyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NHCOOCH₂C₆ H₅ ; R⁴ ═--CH₂ NHCOCH₃

a.N-alpha-(2-Methylsulfonylethyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysine

To a suspension of N-epsilon-benzyloxycarbonyl-L-lysine (20.3 g, 72.25mmol) in DMF (300 ml) was added 2-methylsulfonylethyl p-nitrophenylcarbonate (20.85 g, 72.25 mmol). The reaction mixture was stirredovernight at room temperature, TEA (10.0 ml, 72.25 mmol) was added andthe mixture was stirred for an additional 24 hr. and filtered. Thefiltrate was concentrated under vacuum. The residue was dissolved inEtOAc (300 ml) and washed with aqueous 1N HCl and brine. The organicphase was dried over Na₂ SO₄, filtered and concentrated under vacuum togive a viscous yellow oil. The crude product was purified by flashchromatography on silica gel with CHCl₃, MeOH:CHCl₃ (2:98) and finallyMeOH:CHCl₃ (5:95) to give the product (20.67 g); TLC, R_(f) =0.29,silica gel, MeOH:CHCl₃ :HOAc (9.5:90:0.5).

b.N-alpha-(2-methylsulfonylethylcarbonyl)-N-epsilon-benzyl-oxycarbonyl-L-lysyl-L-valyl-L-prolinet-butyl ester

A solution of the product of Example 14a (2.00 g, 4.65 mmol) in dry THF(25 ml) under a nitrogen atmosphere was cooled to -20° C.N-methylmorpholine (0.51 ml, 4.65 mmol) was added followed by isobutylchloroformate (0.6 ml, 4.65 mmol). The mixture was stirred for 10 minand then cooled to -50° C. A solution of the product of Example 6b (1.25g, 4.65 mmol) in dry THF (25 ml) was added dropwise. The solution wasallowed to warm to room temperature slowly and was stirred overnight.The reaction mixture was filtered and the filtrate concentrated undervacuum. The crude product was purified by flash chromatography on silicagel with MeOH:CHCl₃ (5:95) to give the product (2.47 g); TLC, R_(f)=0.25, silica gel, MeOH:CHCl₃ (5:95).

c.N-alpha-(2-Methylsulfonylethylcarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-proline

Trifluoroacetic acid (2.5 ml, 32.5 mmol) was added to a solution of theproduct of Example 14b (1.0 g, 1.47 mmol) in CH₂ Cl₂ (2.5 ml). Thereaction mixture was stirred for 3.5 hr. and then concentrated undervacuum. The resulting syrup was triturated repeatedly with Et₂ O to givethe product (0.96 g).

d.N-alpha-(2-Methylsulfonylethylcarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

The title compound was prepared as in Example 6j except that the productof Example 14c (0.96 g, 1.53 mmol) in THF (10 ml) was used, andN-methylmorpholine (0.17 ml, 1.53 mmol), isobutyl chloroformate (0.20ml, 1.53 mmol) and L-valinal diethylacetal (0.27 g, 1.53 mmol) in THF(10 ml) were added successively. The crude product was purified by flashchromatography on silica gel with MeOH:CHCl₃ (2.5:97.5) and thenMeOH:CHCl₃ (5:95) to give the product (0.65 g); TLC, R_(f) =0.40, silicagel, MeOH:CHCl₃ (10:90).

e.N-alpha-(2-Methylsulfonylethyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal (Alternate synthesis)

1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.74 g,8.88 mmol) and N-methylmorpholine (1.34 ml, 12.12 mmol) were added to asolution of the product of Example 13c (3.0 g, 8.08 mmol). The productof Example 14a (3.48 g, 8.08 mmol) and HOBT (2.18 g, 16.16 mmol) in CH₂Cl₂ (60 ml) at 0° C. under a nitrogen atmosphere. The reaction mixturewas stirred at 0° C. for 1 hr. and then at room temperature overnight.The reaction mixture was concentrated in vacuo and the residue was takenup in EtOAc, washed with H₂ O, aqueous 1N HCl, brine, 5% aqueous NaHCO₃and brine, dried over MgSO₄. This solution was filtered and concentratedto give the crude product which was purified by flash chromatography onsilica gel with MeOH:CH₂ Cl₂ (5:95) to give the product (5.0 g); TLC,R_(f) =0.60 g, silica gel, MeOH:CH₂ Cl₂ (5:95).

f. N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Aqueous 4N NaOH (0.315 ml) was added to a solution of the product ofExample 14d (0.495 g, 0.63 mmol) in MeOH (6 ml). The reaction mixturewas stirred for 5 min. Aqueous 1N HCl (1.89 ml) was added and thereaction mixture was concentrated under vacuum. The residue wasdissolved in EtOAc (30 ml), washed with aqueous 5% NaHCO₃ and brine,dried over Na₂ SO₄, filtered and concentrated under vacuum to give theproduct (0.395 g); TLC, R_(f) =0.5, silica gel, MeOH:CHCl₃ (10:90).

g.N-Acetylglycyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

N-acetylglycine (0.073 g, 0.62 mmol) and DCC (0.14 g, 0.68 mmol) wereadded to a solution of the product of Example 14f (0.395 g, 0.62 mmol)in DMF (2 ml). The reaction mixture was stirred overnight at roomtemperature. The reaction mixture was filtered and concentrated invacuo. The residue was dissolved in CHCl₃, filtered and concentratedunder vacuum to give the crude product which was purified by flashchromatography on silica gel with MeOH:CHCl₃ (5:95) to give the product(0.37 g); TLC, R_(f) =0.44, silica gel, MeOH:CHCl₃ :HOAc (9.5/90/0.5).

h.N-Acetylglycyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Dowex® 50WX8-H resin (2 ml) was added to a solution of the product ofExample 14g (0.30 g, 0.41 mmol) in acetone: H₂ O (1:4, 10 ml) and thereaction mixture was stirred for 48 hr. The reaction mixture wasfiltered and the filtrate was concentrated under vacuum to give theproduct (0.13 g); TLC, R_(f) =0.35, silica gel MeOH/CHCl₃ (10:90).

EXAMPLE 15N-alpha-(2-Methylsulfonylethyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula I: R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCO₂ CH₂ C₆H₅ ; R⁴ ═--O(CH₂)₂ SO₂ CH₃

p-Toluenesulfonic acid (0.42 g, 2.2 mmol) was added to a solution of theproduct of Example 14d in acetone (450 ml) and the reaction mixture wasstirred for 2 hr. The reaction mixture was concentrated under vacuum.The residue was dissolved in EtOAc (100 ml), washed with aqueous 5%NaHCO₃ and brine, dried over Na₂ SO₄, filtered and concentrated undervacuum to give the product (0.90 g); TLC, R_(f) =0.43, silica gel,MeOH:CHCl₃ (5:95).

Elemental Analysis. Calculated for C₃₃ H₅₁ N₅ O₁₀ S.0.5H₂ O: C, 55.14;H, 7.29; N, 9.74. Found: C, 55.21; H, 7.41; N, 9.24.

EXAMPLE 16 N-Benzyloxycarbonyl-L-norleucyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₃ CH₃ ; R⁴═--OCH₂ C₆ H₅

A mixture of the product of Example 13d (0.75 g, 1.21 mmol) andp-toluenesulfonic acid (0.066 g, 0.35 mmol) in acetone (100 ml) wasstirred for 3 hr. at room temperature.

The mixture was concentrated under vacuum and the residue was dissolvedin EtOAc. This solution was washed successively with 5% aqueous NaHCO₃and brine, dried over MgSO₄, filtered and concentrated to give a glass.Purification by reverse-phase flash chromatography with MeOH:H₂ O (7:3)gave a white powder (0.414 g); TLC, R_(f) =0.5, C₁₈ reverse-phase,MeOH:H₂ O (7:3).

EXAMPLE 17 N-Succinyl-L-norleucyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₃ CH₃ ; R⁴═--(CH₂)₂ COOH

A mixture of the product of Example 13f (1.81 g, 3.02 mmol) and aqueous1N NaOH (3.13 ml) in MeOH:H₂ O (1:1, 56 ml) was stirred for 2 hr. atroom temperature under a nitrogen atmosphere. The MeOH was removed undervacuum. Acetone (30 ml), H₂ O (200 ml), and Dowex® 50WX8-H resin (120ml) were added and the mixture was stirred for 24 hr. The mixture wasfiltered and the resin was washed with acetone. The combined filtrateswere concentrated to remove the acetone and the aqueous solution waslyophilized to give a fluffy white solid (1.41 g).

Elemental Analysis. Calculated for C₂₅ H₄₂ N₄ O₇ : C, 58.80; H, 8.29; N,10.97. Found: C, 58.48; H, 8.29; N, 10.67.

EXAMPLE 18N-alpha-Trimethylacetyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NHCOOCH₂C₆ H₅ ; R⁴ ═--C(CH₃)₃

a.N-alpha-Trimethylacetyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-proline

A mixture of the product of Example 6d (3.54 g, 5.99 mmol) in CH₂ Cl₂(35 ml) and aqueous 1N NaOH (12 ml) was vigorously stirred at 0° C.Trimethylacetyl chloride (0.73 g, 5.92 mmol) was added in one batch. Thereaction mixture was stirred for 15 min at 0° C., then diluted with H₂ O(25 ml) and the pH adjusted to 2 with aqueous 1N HCl. The mixture wasthen extracted with EtOAc. The combined organic layers were washed withbrine, dried over MgSO₄, filtered and concentrated to give the productas an off-white foam (3.39 g); TLC, R_(f) =0.35, silica gel, MeOH:CH₂Cl₂ :HOAc (5:95:1).

b.N-alpha-Trimethylacetyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

HOBT (1.63 g, 12.09 mmol) was added to a solution of the product ofExample 18a (3.39 g, 6.04 mmol) and L-valinal diethylacetal (1.06 g,6.04 mmol) in dry THF (65 ml) at 0° C. under a nitrogen atmosphere andthe mixture was stirred for 15 min. To the resulting solution was added1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.39 g,7.25 mmol) followed by N-methylmorpholine (0.92 g, 9.06 mmol). Themixture was stirred for 1 hr. at 0° C. and then for 24 hr. at roomtemperature. The reaction mixture was concentrated in vacuo and theresulting residue was partitioned between EtOAc and H₂ O. The organiclayer was isolated and washed with aqueous 1N HCl, brine, saturatedaqueous NaHCO₃ and brine then dried over MgSO₄, filtered andconcentrated to give a crude foam. The product was purified by flashchromatography on silica gel with CH₂ Cl₂ :Et₂ O:MeOH (60:40:2) to givea white foam (2.40 g); TLC, R_(f) =0.45 silica gel, CH₂ Cl₂ :Et₂ O:MeOH(60:40:2).

c.N-alpha-Trimethylacetyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

p-Toluenesulfonic acid (0.25 g, 1.3 mm) was added to a solution of theproduct of Example 18b (2.3 g, 3.2 mmol) in acetone (100 ml) at roomtemperature under a nitrogen atmosphere and the mixture was stirred for3 hr. The reaction mixture was then concentrated in vacuo and theresulting residue was dissolved in EtOAc. This solution was washed with5% aqueous NaHCO₃ and brine, dried over MgSO₄, filtered and concentratedto give a white foam. The product was purified by reverse-phasechromatography with MeOH:H₂ O (7:3) to give a white powder (1.78 g);TLC, R_(f) =0.60, reverse-phase C₁₈, MeOH:H₂ O (7:3).

Elemental Analysis. Calculated for C₃₄ H₅₃ N₅ O₇.0.5H₂ O: C, 62.55; H,8.33; N, 10.72. Found: C, 62.17; H, 8.52; N, 10.41.

EXAMPLE 19N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH₂ C₆ H₅ ; R³ ═--(CH₂)₄ NHCOOCH₂ C₆H₅ ; R⁴ ═--(CH₂)₂ CO₂ CH₃

a. N-Benzyloxycarbonyl-L-phenylalanyl-L-proline t-butyl ester

HOBT (2.70 g, 20 mmol) was added to a solution ofN-benzyloxycarbonyl-L-phenylalanine (2.99 g, 10 mmol) in dry DMF (100ml) at 0° C. under a nitrogen atmosphere and the mixture was stirred for15 min. A solution of DCC (2.26 g, 11 mmol) in dry DMF (20 ml) was addedand the mixture was stirred for 5 min. at 0° C. A solution of L-prolinet-butyl ester (1.71 g, 10 mmol) in dry DMF (30 ml) was then added. Themixture was stirred at 0° C. for 1 hr. and at room temperature for anadditional 48 hr. The reaction mixture was filtered and the filtrateconcentrated under vacuum. The resulting residue was mixed with coldEtOAc (50 ml) and filtered. The filtrate was washed successively with20% aqueous citric acid, brine, 5% aqueous NaHCO₃, and brine. Theorganic phase was dried over Na₂ SO₄, filtered and concentrated to givethe product (3.96 g) as a thick yellow syrup; TLC, R_(f) =0.60, silicagel, EtOAc:CHCl₃ (15:85).

b. L-Phenylalanyl-L-proline t-butyl ester

A mixture of the product of Example 19a (3.80 g, 11.9 mmol) and 10% Pd/C(1 g) in EtOH (150 ml) was placed on a Parr apparatus under H₂ (60 psi)and shaken for 9 hr. at room temperature. The mixture was filteredthrough Celite® and the filtrate was concentrated under vacuum to givethe product (2.47 g).

c.N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolinet-butyl ester

HOBT (3.16 g, 23.4 mmol) was added to a solution ofN-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysine (4.45 g,11.7 mmol) in dry THF (50 ml) at 0° C. under nitrogen atmosphere and themixture was stirred for 15 min. A solution of the product of Example 19b(3.16 g, 11.7 mmol) in dry THF (30 ml) was then added and the mixturewas stirred for an additional 5 min. Then DCC (2.66 g, 12.87 mmol) wasadded and the mixture was stirred for 1 hr. at 0° C. and then at roomtemperature for 36 hr. The reaction mixture was filtered and thefiltrate was concentrated under vacuum. The residue was taken up inCHCl₃ (75 ml) and this solution was cooled to 0° C. and filtered. Thefiltrate was washed successively with 20% aqueous citric acid, 5%aqueous NaHCO₃, and brine. The organic phase was dried over Na₂ SO₄,filtered, and concentrated to give the crude product (5.95 g).Purification by flash chromatography on silica gel with MeOH:CH₂ Cl₂(2.5:97.5) gave the product (4.63 g); TLC, R_(f) =0.7, silica gel,MeOH:CH₂ Cl₂ (5:95).

d. N-epsilon-Benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolinetrifluoroacetic acid salt

Trifluoroacetic acid (30 ml) was added to a solution of the product ofExample 19c (4.42 g, 8.74 mmol) in dry CH₂ Cl₂ (30 ml) under nitrogen.The mixture was stirred at room temperature for 5 hr. Toluene (30 ml)was added and the resulting mixture was concentrated under vacuum togive the product (3.96 g); TLC, R_(f) =0.1 silica gel, MeOH:CH₂ Cl₂:HOAc (5:95:0.5).

e.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-proline

A mixture of the product of Example 19d (3.75 g, 6.36 mmol) in CH₂ Cl₂(43 ml) and aqueous 1N NaOH (12.7 ml) was stirred vigorously at 0° C.while 3-carbomethoxypropionyl chloride (0.783 g, 6.36 mmol) was added inone portion. The resulting mixture was stirred vigorously at 0° C. for12 min. The mixture was then diluted with water and adjusted to pH 2with 1N HCl. The organic layer was collected and the aqueous layer wasextracted twice with CH₂ Cl₂. The organic extracts were combined, driedover Na₂ SO₄, filtered and concentrated to give the product (3.46 g);TLC, R_(f) =0.5, silica, CH₃ OH:CH₂ Cl₂ :HOAc (5:95:0.5).

f.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolyl-L-valinaldiethylacetal

Isobutyl chloroformate (0.66 ml, 5.15 mmole) was added to a solution ofthe product of Example 19e (3.41 g, 5.15 mmol) and N-methylmorpholine(0.563 ml, 5.15 mmol) in dry THF (46 ml) at -15° C. under a N₂atmosphere. The reaction mixture was stirred at -15° C. for 30 min. andthen cooled to -40° C. A solution of L-valinal diethylacetal (0.904 g,5.15 mmol) in dry THF (4 ml) was added dropwise. The resulting mixturewas allowed to warm slowly to room temperature and was stirred for 24hr. The reaction mixture was filtered and the filtrate was concentratedunder vacuum to give a residue which was purified by flashchromatography on silica gel with CH₂ Cl₂ :MeOH (97:3) to give theproduct (2.51 g); TLC, R_(f) =0.45, MeOH:CH₂ Cl₂ (5:95).

Elemental Analysis. Calculated for C₄₂ H₆₀ N₅ O₁₀.0.5H₂ O: C, 62.75; H,7.65; N, 8.71. Found: C, 62.82; H, 7.50; N, 8.48.

g.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolyl-L-valinal

A mixture of the product of Example 19f (0.5 g, 0.628 mmol), acetone(100 ml) and p-toluenesulfonic acid monohydrate (0.026 g, 0.125 mmol)was stirred at room temperature under nitrogen for 5 hr. The mixture wasconcentrated under vacuum. The residue was dissolved in EtOAc and washedwith 5% aqueous NaHCO₃. The organic layer was dried over Na₂ SO₄,filtered, and concentrated under vacuum to give the product (0.45 g);TLC, R_(f) =0.31, silica gel, MeOH:CH₂ Cl₂ (5:95).

Elemental Analysis. Calculated for C₃₈ H₅₁ N₅ O₉.0.5H₂ O: C, 62.45; H,7.17; N, 9.58. Found: C, 62.37; H, 7.10; N, 9.42.

EXAMPLE 20N-alpha-[3-(Methylsulfonylaminocarbonyl)propionyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-lysyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ -- NHCOOCH₂C₆ H₅ ; R⁴ ═--(CH₂)₂ CONHSO₂ CH₃

a.N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

A mixture of the product of Example 6j (3.0 g, 4.0 mmol) in MeOH:H₂ O(1:1, 40 ml) and aqueous 1N NaOH (4.4 ml) was stirred at roomtemperature for 1.5 hr. The solution was acidified with aqueous 1N HCl(5 ml) and extracted with EtOAc. The organic solution was dried over Na₂SO₄, filtered and concentrated under vacuum to give the product (2.66 g,0.36 mmol); TLC, R_(f) =0.55, MeOH:CHCl₃ :HOAc (9.5/90/0.5).

b.N-alpha-[3-(Methylsulfonylaminocarbonyl)propionyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

A solution of the product of Example 20a (0.5 g, 0.682 mmol),methanesulfonamide (65 mg, 0.682 mmol), dicyclohexyl carbodiimide (140mg, 0.68 mmol) and 4-dimethylaminopyridine (83 mg, 0.68 mmol) in dry CH₂Cl₂ (15 ml) was stirred overnight at room temperature. Thedicyclohexylurea was removed by filtration and the filtrate concentratedunder vacuum to give the crude product (0.73 g). The product waspurified by flash chromatography on silica gel with MeOH:CHCl₃ (3:97) togive a white solid (0.33 g); TLC, R_(f) =0.4, silica gel, CH₃ OH:CHCl₃(3:97).

c.N-alpha-[3-(Methylsulfonylaminocarbonyl)propionyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A mixture of the product of Example 20b (0.3 g, 0.37 mmol) and Dowex®50WX8-H resin (12.8 ml) in acetone/water (1:5, 24 ml) was stirred atroom temperature for 48 hr. The resin was removed by filtration and thefiltrate concentrated under vacuum to give an aqueous solution which waslyophilized to give the product (0.18 g) as a white solid; TLC, R_(f)=0.2, silica gel, CH₃ OH:H₂ O (5:95).

EXAMPLE 21N-alpha-Succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenylalanyl-L-prolyl-L-valinal

Formula I: R¹ ═--CH(CH₃)₂ ; R² ═--CH₂ C₆ H₅ ; R³ ═--(CH₂)₄ NHCOOCH₂ C₆H₅ ; R⁴ ═--(CH₂)₂ COOH

A mixture of the product of Example 19f (1.0 g, 1.25 mmol) in CH₃ OH:H₂O (1:1, 26 ml) and aqueous 1N NaOH (1.31 ml) was stirred at roomtemperature for 4 hr. The methanol was removed under vacuum to give anaqueous solution (13 ml) ofN-alpha-succinyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-phenyl-alanyl-L-prolyl-L-valinaldiethylacetal which was immediately added to a stirred mixture of Dowel®50WX8-H resin (46.3 g) in water (94 ml) and acetone (11.8 ml). Thismixture was stirred for 48 hr. at room temperature. The mixture wasfiltered and the resin was washed with acetone. The filtrate andwashings were combined, diluted with water (50 ml) and concentratedunder vacuum to give an aqueous solution (100 ml) which was lyophilizedto give the product (0.54 gm) as a white solid; TLC, R_(f) =0.27, silicagel, MeOH:CH₂ Cl₂ (5:95).

EXAMPLE 22N-alpha-Succinyl-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula I. R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOC₆ H₃ Cl₂; R⁴ ═--(CH₂)₂ COOH

a. N-alpha-t-Butyloxycarbonyl-L-lysyl-L-valyl-L-proline t-butyl ester

A mixture of the product of Example 6c (5.0 g, 7.9 mmol) and 10% Pd/C (1g) in ethanol (50 ml) was shaken on a Parr apparatus under hydrogen (60psi) for 5 hr. The reaction mixture was filtered and concentrated undervacuum to give the crude product. This product was purified by flashchromatography on silica gel with MeOH:CHCl₃ (10:90) to give a whitefoam (4.5 g); TLC, R_(f) =0.3, silica gel, CHCl₃ :MeOH (90:10).

b.N-alpha-t-Butyloxycarbonyl-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-prolinet-butyl ester

2,4-Dichlorobenzoyl chloride (1.87 g, 8.9 mmol) was added to a solutionof the product of Example 22a (4.52 g, 8.9 mmol) and N-methylmorpholine(1.0 g, 10 mmol) in CH₂ Cl₂ (50 ml) at 5° C. The resulting mixture wasstirred for 1 hr. at 5° C. and overnight at room temperature. Themixture was diluted with CH₂ Cl₂ (200 ml) and washed successively with20% citric acid, saturated NaHCO₃ and brine. The organic layer was driedover Na₂ SO₄, filtered and concentrated under vacuum and the product waspurified by flash chromatography on silica gel with MeOH:CHCl₃ (5:95) togive a white foam (2.2 g); TLC, R_(f) =0.6, silica gel, MeOH:CHCl₃(5:95).

c. N-epsilon-(2,4-Dichlorobenzoyl)-L-lysyl-L-valyl-L-prolinetrifluoroacetic acid salt

A mixture of trifluoroacetic acid (7.3 g, 6.4 mmol) and the product ofExample 22b (2.2 g, 3.7 mmol) in CH₂ Cl₂ (5 ml) was stirred for 5 hr. atroom temperature. The mixture was concentrated under vacuum and theresidue was triturated with Et₂ O. The solid product was isolated byfiltration and dried under vacuum to give a white solid (1.73 g).

d.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-proline

A vigorously stirred solution of the product of Example 22c (1.73 g, 2.7mmol) in CH₂ Cl₂ (10 ml) at 5° C. was treated with aqueous 1N NaOH (2.7ml). 3-Carbomethoxypropionyl chloride (0.41 g, 2.7 mmol) was added andthe solution stirred for an additional 15 min. The reaction mixture wasdiluted with H₂ O (50 ml) and the pH was adjusted to 2 with aqueous 1NHCl. The organic phase was separated and the aqueous phase was extractedtwice with CH₂ Cl₂. The combined organic phases were dried over Na₂ SO₄and concentrated under vacuum to give the product (1.32 g) as a paleyellow foam; TLC, R_(f) =0.10, silica gel, MeOH:CHCl₃, (5:95).

e.N-alpha-(3-Carbomethoxypropionyl)-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Isobutyl chloroformate (0.29 g, 2.0 mmol) was added to a solution of theproduct of Example 22d (1.32 g, 2.0 mmol) and N-methylmorpholine (0.210g, 2.0 mmol) in CH₂ Cl₂ (15 ml) at -10° C. under a nitrogen atmosphere.The reaction mixture was stirred at -10° C. for 30 min. and then cooledto -40° C. and L-valinal diethylacetal (0.37 g, 2.0 mmol) was added inone portion. The mixture was stirred at -40° C. for 1 hr. and then atroom temperature for 2 hr. The mixture was concentrated under vacuum andthe product was purified by flash chromatography on silica gel withMeOH:CHCl₃ (3:97) to give a white solid (980 mg); TLC, R_(f) =0.7,silica gel, MeOH:CHCl₃ (5:95).

f.N-alpha-Succinyl-N-epsilon-(2,4-dichlorobenzoyl)-L-lysyl-L-valyl-L-prolyl-L-valinal

A mixture of the product of Example 22e (970 mg, 1.2 mmol) in H₂ O:MeOH(1:1, 30 ml) and aqueous 1N NaOH (1.2 ml) was stirred at roomtemperature for 1.5 hr. The methanol was removed under vacuum. Dowex®50WX8-H resin (50 ml), H₂ O (100 ml) and acetone (20 ml) were added andthe mixture was stirred at room temperature for 24 hr. The reactionmixture was filtered and the resin was washed with acetone. The combinedfiltrates were concentrated under vacuum to remove acetone and theaqueous solution was lyophilized to give the product (700 mg) as a whitesolid; TLC R_(f) =0.4, silica gel, HOAc:MeOH:CHCl₃ (0.25:10:89.75).

EXAMPLE 23N-alpha-(2,4-Dichlorobenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCO₂ --CH₂C₆ H₅ ; R₄ ═--C₆ H₃ Cl₂

a.N-alpha-(2,4-Dichlorobenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

2,4-Dichlorobenzoyl chloride (0.132 g, 0.623 mmol) was added to astirred mixture of the product of Example 14f (0.40 g, 0.632 mmol) inCH.sub. Cl₂ (15 ml) and N-methylmorpholine (0.5 ml) at 0° C. under anitrogen atmosphere. The reaction mixture was stirred for 30 min. andthen poured into aqueous 1N HCl and extracted with EtOAc. The combinedorganic extracts were washed with brine, dried over MgSO₄, filtered andconcentrated to give the product. This oil was purified by flashchromatography on silica gel with MeOH:CH₂ Cl₂ (3:97) to give acolorless oil (0.41 g); TLC, R_(f) =0.60, silica gel, MeOH:CH₂ Cl₂(5:95).

b.N-alpha-2,4-Dichlorobenzoyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The product of Example 23a was treated with p-toluenesulfonic acid asdescribed in Example 10d to give a white foam (0.34 g); TLC, R_(f) =0.5,silica gel, CH₂ Cl₂ :MeOH (95:5).

Elemental Analysis. Calculated for C₃₆ H₄₇ N₅ Cl₂ O₇.H₂ O: C, 57.59; H,6.57; N, 9.32. Found: C, 57.75; H, 6.50; N, 9.06.

EXAMPLE 24N-alpha-(4-Carboxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-propyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ --NHCOCH₂ C₆H₅ ; R⁴ ═--C₆ H₄ CO₂ H

a.N-alpha-(4-Carbomethoxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.133 g,0.69 mmol) and N-methylmorpholine (0.1 ml, 0.95 mmol) were added to asolution of the product of Example 14f (0.40 g, 0.632 mmol),p-carbomethoxy benzoic acid (0.114 g, 0.632 mmol) and HOBT (0.17 g, 1.26mmol) in dry THF (20 ml) at 0° C. under a N₂ atmosphere. The reactionmixture stirred at 0° C. for 1 hr. and then at room temperatureovernight. The reaction mixture was concentrated under vacuum. Theresidue was taken up in EtOAc washed with aqueous 1N HCl, brine, 5%aqueous NaHCO₃ and brine, dried over MgSO₄, filtered and concentrated togive the crude product. Purification by flash chromatography on silicagel with CH₂ Cl₂ :MeOH (95:5) gave a white foam (0.32 g); TLC, R_(f)=0.6, silica gel, CH₂ Cl₂ :MeOH (95:5).

b.N-alpha-(4-Carboxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

1N NaOH (0.5 mmol) was added to the product of Example 24a (0.32 g, 0.40mmol) in MeOH (5 ml) and H₂ O (3 ml) at room temperature under N₂ andthe mixture was stirred overnight. The MeOH was removed in vacuo,acetone (10 ml) and 1N HCl (10 ml) were added and the mixture wasstirred overnight. The acetone was removed under vacuum and the aqueouslayer was extracted repeatedly with EtOAc. The combined organic extractswere washed with brine, dried over MgSO₄ and concentrated to give thecrude product. Purification by flash chromatography on silica withMeOH:CH₂ Cl₂ :HOAc (5:95:0.5) gave a white foam (0.210 g); TLC, R_(f)=0.65, silica gel, MeOH:CH₂ Cl₂ :HOAc (10:90:1).

EXAMPLE 25N-alpha-(2-Carboxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOCH₂ --C₆H₅ ; R⁴ ═--C₆ H₄ CO₂ H

a.N-alpha-(2-Carboxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

Phthalic anhydride (0.117 g, 0.79 mmol) was added to a solution of theproduct of Example 14f (0.50 g, 0.79 mmol) in acetonitrile (10 ml). Thereaction mixture was stirred overnight at room temperature thanconcentrated under vacuum to give the crude product. This compound waspurified by flash chromatography on silica gel with CH₂ Cl₂ :MeOH:HOAc(95:5:1) to give an oil (0.42 g); TLC, R_(f) =0.5, silica gel, CH₂ Cl₂:MeOH:HOAc (95:5:1).

b.N-alpha-(2-Carboxybenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The product of Example 25a was reacted with p-toluenesulfonic acid asdescribed in Example 10d. The product was purified by flashchromatography on a reverse-phase C₁₈ column with THF:H₂ O (1:1) to givea white foam (0.15 g); TLC, R_(f) =0.75, reverse-phase (C₁₈), THF:H₂ O(1:1).

EXAMPLE 26N-alpha-(4-Phenylbenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCO₂ --CH₂C₆ H₅ ; R⁴ ═--C₆ H₄ C₆ H₅

a.N-alpha-(4-Phenylbenzoyl)-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (0.133 g,0.69 mmol) and N-methylmorpholine (0.1 ml, 0.95 mmol) were added to asolution of the product of Example 14f (0.40 g, 0.63 mmol),4-phenylbenzoic acid (0.125 g, 0.63 mmol) and HOBT (0.171 g, 1.26 mmol)in CH₂ Cl₂ (20 ml) at 0° C. under a N₂ atmosphere. The reaction mixturewas stirred at 0° C. for 1 hr. and then at room temperature overnight.The reaction mixture was poured into aqueous 1N HCl and the organiclayer was isolated and washed with brine, 5% aqueous NaHCO₃ and brine,dried over MgSO₄, filtered, then concentrated to give the crude product.Purification by flash chromatography on silica gel with CH₂ Cl₂ :MeOH(95:5) gave a white foam (0.37 g); TLC, R_(f) =0.55, silica gel, CH₂ Cl₂:MeOH (95:5).

b.N-alpha-4-Phenylbenzoyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The product of Example 26a was treated with p-toluenesulfonic acid asdescribed in Example 10d to give a white foam (0.31 g); TLC, R_(f)=0.35, silica gel, MeOH:CH₂ Cl₂ (5:95).

Elemental Analysis. Calculated for C₄₂ H₅₃ N₅ O₇.0.5H₂ O: C, 67.35; H,7.26; N, 9.35. Found: C, 67.11; H, 7.43; N, 9.16.

EXAMPLE 27 N-alpha-[2-(2-Pyridyl)-ethyloxycarbonyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula I: R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCO₂ CH₂ C₆H₅ ; R⁴ ═--O(CH₂)₂ C₅ H₄ N

a.N-alpha-[2-(2-Pyridyl)-ethyloxycarbonyl]-N-epsilon-benzyloxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

To a stirred solution of the product of Example 14f (400 mg, 0.63 mmol)in acetonitrile:H₂ O (40 ml, 1:1) was added 2-(2-pyridyl)-ethylp-nitrophenyl carbonate hydrochloride (208 mg, 0.64 mmol). Whendissolution was complete, triethylamine (127 mg, 1.27 mmol) was addedand the mixture was stirred at room temperature for 2 hr. Theacetonitrile was removed under vacuum and the aqueous phase was dilutedwith water (20 ml) and extracted with EtOAc (2×200 ml). The combinedextracts were dried (Na₂ SO₄) and concentrated to yield the crudeproduct. Purification by flash chromatography on silica gel withMeOH:CHCl₃ (5:95) gave the title compound (250 mg) as a white foam.

Elemental Analysis. Calculated for C₄₁ H₆₂ N₆ O₉ : C, 62.89; H, 7.98; N,10.73. Found: C, 63.01; H, 8.07; N, 10.70.

b.N-alpha-[2-(2-Pyridyl)-ethyloxycarbonyl]-N-epsilon-benzyl-oxycarbonyl-L-lysyl-L-valyl-L-prolyl-L-valinal

A mixture of the product of Example 27a (240 mg, 0.31 mmol) andp-toluenesulfonic acid (117 mg, 0.62 mmol) in acetone (20 ml) wasstirred for 3 hr. at room temperature. The solvent was removed undervacuum and the residue was dissolved in EtOAc. This solution was washedwith aqueous 1N NaHCO₃ and dried (Na₂ SO₄). Evaporation of the solventgave a white foam (226 mg); TLC, R_(f) =0.30, silica gel, MeOH:CHCl₃(6:94).

Elemental Analysis. Calculated for C₃₇ H₅₂ N₆ O₈.H₂ O: C, 61.14; H,7.48; N, 11.56. Found: C, 60.71; H, 7.44; N, 11.43.

EXAMPLE 28N-alpha-N-epsilon-Dibenzoyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOC₆ H₅ ;R⁴ ═--C₆ H₅

a. N-alpha-N-epsilon-Dibenzoyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (0.225 g,1.18 mmol) and N-methylmorpholine (0.17 ml, 1.6 mmol) were added to asolution of the product of Example 13c (0.40 g, 1.07 mmol),N-alpha-N-epsilon dibenzoyl L-lysine (0.388 g, 1.07 mmol) and HOBT(0.289 g, 2.14 mmol) in CH₂ Cl₂ (25 ml) at 0° C. under N₂ atmosphere.The reaction mixture was stirred at 0° C. for 1 hr. and then at roomtemperature overnight. The reaction mixture was concentrated and theoily residue was dissolved in EtOAc, washed with aqueous 1N, HCl, brine,5% aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated to givethe crude product. This material was purified by flash chromatography onsilica gel with CH₂ Cl₂ :MeOH (95:5) to give a white foam (0.58 g); TLC,R_(f) =0.55, silica gel, CH₂ Cl₂ :MeOH (95:5).

b. N-alpha-N-epsilon-Dibenzoyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The product of Example 28a was reacted with p-toluenesulfonic acid asdescribed in Example 10d to give a white foam (0.56 g); TLC, R_(f)=0.45, silica gel, CH₂ Cl₂ :MeOH (95:5).

Elemental Analysis. Calculated for C₃₅ H₄₇ N₅ O₆.0.5H₂ O: C, 65.39; H,7.52; N, 10.89. Found: C, 65.27; H, 7.58; N, 10.71.

EXAMPLE 29 N-alpha-N-epsilon-Diacetyl-L-lysyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂ ; R² ═--CH(CH₃)₂ ; R³ ═--(CH₂)₄ NHCOCH₃ ; R⁴═--CH₃

a. N-alpha-N-epsilon-Diacetyl-L-lysyl-L-valyl-L-prolyl-L-valinaldiethylacetal

The title compound was prepared as in Example 28a, using1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (0.285 g,1.5 mmol), N-methylmorpholine (0.22 ml, 2.03 mmol), the product ofExample 13c (0.50 g, 1.35 mmol), N-alpha-N-epsilon-diacetyl-L-lysine(0.308 g, 1.35 mmol) and HOBT (0.365, 2.7 mmol) in THF (15 ml) and DMF(7 ml). The product was purified by flash chromatography with CH₂ Cl₂:MeOH (9:1) to give an off-white foam (0.38 g); TLC, R_(f) =0.6, silicagel, CH₂ Cl₂ :MeOH (9:1).

b. N-alpha-N-epsilon-Diacetyl-L-lysyl-L-valyl-L-prolyl-L-valinal

The product of Example 29a was treated with p-toluenesulfonic acid asdescribed in Example 10d to give an off-white foam (0.29 g); TLC, R_(f)=0.5, silica gel, CH₂ Cl₂ :MeOH (85:15).

Elemental Analysis. Calculated for C₂₅ H₄₃ N₅ O₆.1.25H₂ O: C, 56.42; H,8.61; N, 13.16. Found: C, 56.42; H, 8.51; N, 12.86.

EXAMPLE 30 N-Benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-valinal

Formula (I): R¹ ═--CH(CH₃)₂, R² ═--CH(CH₃)₂ ; R³ ═--CH₂ C₆ H₅, R⁴═--OCH₂ C₆ H₅

a. N-Benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-valinaldiethylacetal

The title compound was prepared as in Example 1e, using a solution ofN-benzyloxycarbonyl-L-phenlalanine (0.40 g, 1.35 mmol) andN-methylmorpholine (0.15 ml, 1.35 mmol) in dry THF (10 ml) and addingisobutyl chloroformate (0.17 ml, 1.35 mmol) followed by a solution theproduct of Example 13c (0.50 g, 1.35 mmol) in dry THF (10 ml). The crudeproduct was purified by flash chromatography on silica gel withMeOH:CHCl₃ (2:98) to give the product (0.78 g); TLC, R_(f) =0.41, silicagel, MeOH:CHCl₃ (5:95).

b. N-Benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-prolyl-L-valinal

p-Toluenesulfonic acid (160 mg, 0.84 mmol) was added to a solution ofthe product of Example 30a (0.38 g, 0.58 mmol) in acetone (100 ml) andthe reaction was stirred for 2 hr. The reaction was concentrated undervacuum and the residue was dissolved in EtOAc (50 ml). This solution waswashed with aqueous 5% NaHCO₃ and brine, dried over Na₂ SO₄, filteredand concentrated under vacuum to give the product (0.29 g); TLC, R_(f)=0.29, silica gel, MeOH:CHCl₃ (5:95).

Elemental Analysis. Calculated for C₃₂ H₄₂ N₄ O₆.0.5H₂ O: C, 65.40; H,7.37; N, 9.53. Found: C, 65.76; H, 7.46; N, 9.22.

EXAMPLE 31 Preparation of Bisulfite Adducts of Formula (I) Aldehydes ofthe Invention

A solution of an aldehyde of the invention in MeOH:H₂ O (2:1 to 1:1,5-10 ml/g of aldehyde) was treated with 2 equivalents of NaHSO₃ andstirred until dissolution was complete. The MeOH was removed undervacuum and the resulting aqueous solution was lyophilized to give theproduct as a white solid.

Bisulfite adducts of Formula (I) aldehydes prepared in this manner areshown in Table I.

                                      TABLE I                                     __________________________________________________________________________    Bisulfite Adducts of Formula I Compounds                                      R.sup.1 = R.sup.2 = --CH(CH.sub.3).sub.2                                      Aldehyde                                                                      from             Molecular Elemental Analysis                                 Example                                                                            R.sup.3                                                                         R.sup.4   Composition                                                                             Calculated                                                                           Found                                       __________________________________________________________________________     1f  b --OC(CH.sub.3).sub.3                                                                    C.sub.34 H.sub.53 N.sub.5 O.sub.8 --                                                    C, 45.18                                                                             45.16                                                        .2NaHSO.sub.3 2H.sub.2 O                                                                H, 6.58                                                                              6.21                                                                   N, 7.75                                                                              7.64                                                                   S, 7.09                                                                              7.84                                         4d  a --OC(CH.sub.3).sub.3                                                                    C.sub.33 H.sub.52 N.sub.6 O.sub.7 --                                                    C, 44.59                                                                             44.81                                                        .2NaHSO.sub.3.2H.sub.2 O                                                                H, 6.58                                                                              6.46                                                                   N, 9.45                                                                              9.05                                         6k  b --(CH.sub.2).sub.2 CO.sub.2 H                                                           C.sub.33 H.sub.49 N.sub.5 O.sub.9 --                                                    C, 44.67                                                                             45.69                                                        .2NaHSO.sub.3                                                                           H, 5.92                                                                              5.88                                                                   N, 8.07                                                                              7.88                                         8b  b --(CH.sub.2).sub.2 CO.sub.2 CH.sub.3                                                    C.sub.34 H.sub.51 N.sub.5 O.sub.9--                                                     C, 44.49                                                                             44.62                                                        .2NaHSO.sub.3.2H.sub.2 O                                                                H, 6.26                                                                              5.96                                                                   N, 7.63                                                                              7.70                                                                   S, 6.99                                                                              7.39                                        10d  b --OCH.sub.2 C.sub.6 H.sub.5                                                             C.sub.37 H.sub.51 N.sub.5 O.sub.8 --                                                    C, 49.27                                                                             49.34                                                        .2NaHSO.sub.3                                                                           H, 5.92                                                                              5.98                                                                   N, 7.76                                                                              7.58                                        11f  c --(CH.sub.2).sub.2 CO.sub.2 H                                                           C.sub.25 H.sub.42 N.sub.4 O.sub.7 --                                                    C, 39.78                                                                             39.30                                                        .2NaHSO.sub.3.2H.sub.2 O                                                                H, 6.41                                                                              5.89                                                                   N, 7.42                                                                              7.14                                        13g  d --(CH.sub.2).sub.2 CO.sub.2 CH.sub.3                                                    C.sub.26 H.sub.44 N.sub.4 O.sub.7 --                                                    C, 42.61                                                                             43.69                                                        .2NaHSO.sub.3                                                                           H, 6.32                                                                              6.43                                                                   N, 7.64                                                                              7.67                                        14h  b --CH.sub.2 NHCOCH.sub.3                                                                 C.sub.33 H.sub.50 N.sub.6 O.sub.8 --                                                    C, 43.90                                                                             43.28                                                        .2NaHSO.sub.3.2H.sub.2 O                                                                H, 6.25                                                                              5.86                                                                   N, 9.31                                                                              8.94                                        18c  b --C(CH.sub.3).sub.3                                                                     C.sub.34 H.sub.53 N.sub.5 O.sub.7 --                                                    C, 47.93                                                                             47.83                                                        .2NaHSO.sub.3                                                                           H, 6.50                                                                              6.46                                                                   N, 8.22                                                                              8.14                                        23b  b C.sub.6 H.sub.3 Cl.sub.2                                                                C.sub.36 H.sub.47 N.sub.5 Cl.sub.2 O.sub.7 --                                           C, 45.09                                                                             45.02                                                        .2NaHSO.sub.3.H.sub.2 O                                                                 H, 5.36                                                                              5.35                                                                   N, 7.30                                                                              6.99                                        26b  b C.sub.6 H.sub.4 C.sub.6 H.sub.5                                                         C.sub.42 H.sub.53 N.sub.5 O.sub.6 --                                                    C, 53.21                                                                             53.35                                                        .2NaHSO.sub.3                                                                           H, 5.85                                                                              5.97                                                                   N, 7.38                                                                              7.41                                        b    e C.sub.6 H.sub.5                                                                         C.sub.35 H.sub.47 N.sub.5 O.sub.7 --                                                    C, 48.90                                                                             49.14                                                        .2NaHSO.sub.3.H.sub.2 O                                                                 H, 5.97                                                                              5.86                                                                   N, 8.14                                                                              8.03                                        __________________________________________________________________________     Notes:                                                                        a  R.sup.3 = --(CH.sub.2).sub.4 NHCONHC.sub. 6 H.sub.5                        b  R.sup.3 = --(CH.sub.2).sub.4 NHCO.sub.2 CH.sub.2 C.sub.6 H.sub.5           c  R.sup.3 = --CH.sub.2 CH(CH.sub.3).sub.2                                    d  R.sup.3 = --(CH.sub.2).sub.3 CH.sub.3                                      e  R.sup.3 = --(CH.sub.2).sub.4 NHCOC.sub.6 H.sub.5                      

EXAMPLE 32N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-prolyl-L-valinal

Formula (II): R⁵ ═--CH(CH₃)₂ ; R⁶ ═--CH(CH₃)₂ ; R⁷ ═--CH₂ CH(CH₃)₂ ; R⁸═--OCH₃.

a. N-alpha-t-Butyloxycarbonyl-L-valyl-L-proline benzyl ester

The title compound was prepared as in Example 1a, using a solution ofN-alpha-t-butyloxycarbonyl-L-valine (5.0 g, 23 mmol) in DMF (80 ml)cooled to 0° C. and adding successively DCC (5.15 g, 25 mmol), HOBT(6.21 g, 46 mmol) and a slurry of L-proline benzyl ester (5.56 g, 23mmol) in TEA (3.4 ml, 23 mmol) and DMF (20 ml). The crude yellow oil wasdissolved in EtOAc (200 ml) and the solution washed successively with20% aqueous citric acid (200 ml), saturated aqueous NaHCO₃, aqueous 1NHCl and brine. The organic layer was dried over Na₂ SO₄, filtered andconcentrated under vacuum to give the crude product as a yellow oil.Purification by flash chromatography on silica gel with EtOAc:CH₂ Cl₂(5:95) followed by EtOAc:CH₂ Cl₂ (1:9) gave the product (7.36 g); TLC,R_(f) =0.57, silica gel, EtOAc:CH₂ Cl₂ (1:4).

b. L-Valyl-L-proline benzyl ester hydrochloride

The title compound was prepared as in Example 1b using a solution of theproduct of Example 32a (5.0 g, 12.4 mmol) in EtOAc (20 ml) and adding 6NHCl/EtOAc (20 ml) to give a solid residue which was triturated withpetroleum ether and filtered under N₂ to yield the purified product (4.1g) as a white solid.

c. L-Leucine methyl ester isocyanate

A three-neck 1 liter round bottom flask, fitted with gas inlet,mechanical stirrer, reflux condenser and gas outlet connected to a 30%aqueous NaOH trap was charged with L-leucine methyl ester hydrochloride(61.0 g, 0.336 mmol) and dry toluene (250 ml). The mixture was heated togentle reflux for 0.5 hr. while a vigorous stream of phosgene wascontinuously introduced. Following dissolution of the solids (0.5 hr.)phosgene was introduced for an additional 15 min. The product wasisolated by initially removing the toluene under vacuum and fractionallydistilling the remaining residue under vacuum to give the product (48.0g), bp=71°-73° C. at 0.8 torr.

d. N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-proline benzylester

N-Methylmorpholine (6.0 ml, 54.5 mmol) and a solution of the product ofExample 32c (10.0 g, 58.5 mmol) in CHCl₃ (50 ml) was added to a solutionof the product of Example 32b (18.57 g, 54.5 mmol) in CHCl₃ (250 ml)cooled to 0° C. The solution was stirred for 2 hr. and the solventremoved under vacuum. The residue was dissolved in EtOAc (200 ml) andwashed successively with saturated aqueous NaHCO₃, H₂ O, aqueous 1N HCl,and brine. The organic layer was dried with Na₂ SO₄, filtered andconcentrated under vacuum to give the crude product (27.54 g) as a whitefoam. Purification using a Waters Prep 500® Liquid Chromatograph withhexane:Et₂ O (1:3) as eluent gave the desired product (20.87 g); TLC,R_(f) =0.57, silica gel, Et₂ O.

Elemental Analysis. Calculated for C₂₅ H₃₇ N₃ O₆ : C, 63.14; H, 7.84; N,8.84. Found: C, 63.32; H, 7.99; N, 8.59.

e. N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-proline

A solution of the product of Example 32d (5.16 g) and 10% Pd/C (1.0 g)in ethanol (100 ml) was hydrogenated using a Parr shaker (3 atm. H₂) for3 hr. The reaction mixture was filtered through Celite® and the filtrateconcentrated under vacuum to give a gummy residue which was trituratedwith hexane to yield the product (3.54 g) as a white solid.

Elemental Analysis. Calculated for C₁₈ H₃₁ N₃ O₆ : C, 56.09; H, 8.11; N,10.90 . Found: C, 55.51; H, 7.91; N, 10.68.

f.N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-prolyl-L-valinol

The title compound was prepared as in Example 1e using a solution of theproduct of Example 32e (2.0 g, 5.2 mmol) in dry THF (30 ml) and addingN-methylmorpholine (0.57 ml, 5.2 mmol), isobutyl chloroformate (0.68 ml,5.2 mmol) and a solution of L-valinol (0.535 g, 5.2 mmol) in dry THF (10ml) to give the crude product (2.38 g) which was used without furtherpurification; TLC, R_(f) =0.44, silica gel, MeOH:CHCl₃ (1:9).

g.N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-prolyl-L-valinal

The title compound was prepared as in Example 1f using oxalyl chloride(0.25 ml, 2.85 mmol), in CH₂ Cl₂ (4 ml) and adding DMSO (0.4 ml, 5.7mmol) in CH₂ Cl₂ (1 ml), the product of Example 32f (0.9 g, 1.9 mmol) inCH₂ Cl₂ (2 ml) and TEA (0.8 ml, 5.7 mmol). The crude mixture waspurified using three successive flash column chromatographies on silicagel with MeOH:CHCl₃ (5:95), MeOH:CHCl₃ (2.5:97.5), and EtOAc:Et₂ O (1:1)respectively as eluents to give the product (0.625 g); TLC, R_(f) =0.37,silica gel, MeOH:CHCl₃ (5:95).

Elemental Analysis. Calculated for C₂₃ H₄₀ N₄ O₆ : C, 58.95; H, 8.60; N,11.96. Found: C, 58.79; H, 8.77; N, 12.14.

h.N-[1(S)-Carbomethoxy-3-methylbutyl]carbamoyl-L-valyl-L-prolyl-L-valinalbisulfite

A solution of the product of Example 32g (0.515 g, 1.1 mmol) and NaHSO₃(0.229 g, 2.2 mmol) in MeOH:H₂ O (1:1) (10 ml) was stirred at roomtemperature until dissolution was complete. The product (0.7 g) wasisolated by removing the MeOH under vacuum and the H₂ O using alyophilizer.

Elemental Analysis. Calculated for C₂₃ H₄₀ N₄ O₆.2NaHSO₃.3H₂ O: C,39.76; H, 6.38; N, 8.07. Found: C, 39.89; H, 6.22; N, 8.06.

EXAMPLES 33-35 Formula (II): R³ ═see table; R⁶ ═--CH(CH₃)₂ ; R⁷ ═CH₂CH(CH₃)₂ ; R⁸ ═--OCH₃.

In a manner analogous to that described in Example 32, steps f and g,the product of Example 32e was converted to the compounds of Table II byuse of the appropriate amino alcohol containing the substituent R⁵.

                                      TABLE II                                    __________________________________________________________________________                            Elemental Analysis                                    Example                                                                            R.sup.5    Formula Calc'd                                                                              Found                                                                              R.sub.f                                    __________________________________________________________________________    33   --CH(CH.sub.3)CH.sub.2 CH.sub.3                                                          C.sub.24 H.sub.42 N.sub.4 O.sub.6                                                     C, 58.63                                                                            58.59                                                                              0.51                                                       0.5H.sub.2 O                                                                          H,  8.82                                                                             8.47                                                                   N, 11.40                                                                            11.47                                           34   D,L-(CH.sub.2).sub.2 CH.sub.3                                                            C.sub.23 H.sub.40 N.sub.4 O.sub.6                                                     C, 56.77                                                                            56.48                                                                              0.49                                                       .H.sub.2 O                                                                            H,  8.69                                                                             8.44                                                                   N, 11.51                                                                            11.20                                           35   --(CH.sub.2).sub.2 CH.sub.3                                                              C.sub.23 H.sub.40 N.sub.4 O.sub.6                                                     C, 56.77                                                                            57.15                                                                              0.48                                                       .H.sub.2 O                                                                            H,  8.69                                                                             8.34                                                                   N, 11.51                                                                            11.50                                           __________________________________________________________________________

All R_(f) values in Table II were obtained by TLC on silica gel, usingEtOAc as eluent.

EXAMPLE 36N-alpha-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinal

Formula (III): R⁹ ═--CH(CH₃)₂ ; R¹⁰ ═--(CH₂)₄ NHCOOCH₂ C₆ H₅ ; R¹¹═--OC(CH₃)₃.

a.N-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolinemethyl ester

Isobutyl chloroformate (0.13 ml, 1.0 mmol) was added dropwise to asolution ofN-alpha-t-butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysine andN-methylmorpholine (0.12 ml, 1.1 mmol) in THF (8.0 ml) cooled to -23° C.under N₂. The reaction mixture was stirred for 20 min. and the solutioncooled to -50° C. A solution of L-proline methyl ester hydrochloride andN-methylmorpholine (0.12 ml, 1.1 mmol) in DMF (2 ml) was added in oneportion and the reaction mixture was allowed to warm slowly to roomtemperature and was stirred overnight. The solution was filtered,concentrated under vacuum and the residue dissolved in EtOAc (100 ml).The EtOAc solution was washed with 1N aqueous HCl, saturated NaHCO₃, andbrine. The organic layer was dried over Na₂ SO₄, filtered andconcentrated under vacuum to give the crude product which was purifiedby flash chromatography using Et₂ O as the eluent to give the finalproduct (0.36 g); TLC, R_(f) =0.44, silica gel, Et₂ O.

b. N-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-proline

A solution of the product of Example 36a (0.36 g, 0.73 mmol) in MeOH(2.5 ml) and 1N aqueous NaOH (1.0 ml) was stirred at room temperaturefor 5 hr. The solution was concentrated under vacuum and the residuedissolved in H₂ O (10 ml) and extracted with Et₂ O. The aqueous layerwas acidified with 10% citric acid whereupon a white solid precipitated.The solid was extracted into EtOAc and the organic layer dried with Na₂SO₄ and concentrated under vacuum to give the product (0.25 g); TLC,R_(f) =0.7, silica gel, MeOH:CHCl₃ :HOAc (9.5:90:0.5).

c.N-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valino

Isobutyl chloroformate (70 ml, 0.52 mmol) was added dropwise to asolution of the product of Example 36b (0.25 g, 0.52 mmol) andN-methylmorpholine (60 ml, 0.55 mmol) in THF (6 ml) cooled to -20° C.under a nitrogen atmosphere. The reaction mixture was stirred for 15min. and the solution cooled to -45° C. A solution of L-valinol (0.054g, 0.52 mmol) in THF (2 ml) was added, the reaction mixture stirred for2 hr. and the solution was allowed to warm to room temperature and wasstirred overnight. The reaction mixture was concentrated under vacuumand the residue dissolved in EtOAc. The solution was washed successivelywith 1N aqueous HCl, saturated NaHCO₃ and brine. The organic layer wasdried with Na₂ SO₄, filtered and concentrated under vacuum to give theproduct (0.26 g) as a white foam; TLC, R_(f) =0.45, silica gel,MeOH:CHCl₃ (5:95).

d.N-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinal

The title compound was prepared as in Example 1f using oxalyl chloride(0.44 g, 3.5 mmol) in CH₂ Cl₂ (40 ml) and successively adding DMSO (0.68g, 8.75 mmol) in CH₃ Cl₂ (7 ml), the product of Example 36c (0.90 g, 1.6mmol) in CH₂ Cl₂ (7 ml) and TEA (0.68 g, 6.73 mmol) to give the crudeproduct. Purification by flash chromatography on silica gel withMeOH:Et₂ O (4:96) gave the product (1.50 g); TLC, R_(f) =0.54, silicagel, MeOH:Et₂ O (4:96).

Elemental Analysis. Calculated for C₂₉ H₄₄ N₄ O₇.H₂ O: C, 60.19; H,8.01; N, 9.68. Found: C, 60.40; H, 7.68; N, 9.60.

e.N-t-Butyloxycarbonyl-N-epsilon-benzyloxycarbonyl-L-lysyl-L-prolyl-L-valinalbisulfite

A solution of the product of Example 36d (1.51 g, 2.69 mmol) and NaHSO₃(0.59 g, 5.65 mmol) in MeOH (15 ml) and H₂ O (8 ml) was stirred untildissolution was complete. The MeOH was removed under vacuum and the H₂ Oremoved with a lyophilizer to give the product (1.75 g).

Elemental Analysis. Calculated for C₂₉ H₄₄ N₄ O₇.2NaHSO₃ : C, 45.31; H,6.03; N, 7.29. Found: C, 45.45; H, 5.87; N, 7.30.

EXAMPLE 37 N-t-Butyloxycarbonyl-L-valyl-L-prolyl-L-valinal

Formula (III): R⁹ ═--CH(CH₃)₂ ; R¹⁰ ═--CH(CH₃)₂ ; R¹¹ ═--OC(CH₃)₃.

a. N-t-Butyloxycarbonyl-L-valyl-L-proline

A mixture of the product of Example 32a (7.25 g, 17.9 mmol) and 10% Pd/C(1.4 g) in ethanol (150 ml) was stirred under 3 atmospheres of H₂ atroom temperature for 2 hr. The reaction mixture was filtered throughCelite® and the filtrate concentrated under vacuum to give the desiredproduct (5.68 g).

b. N-t-Butyloxycarbonyl-L-valyl-L-prolyl-L-valinol

Isobutyl chloroformate (1.25 ml, 9.6 mmol) was added to a solution ofthe product of Example 37a (3.0 g, 9.6 mmol) and N-methylmorpholine(1.05 ml, 9.6 mmol) in THF (40 ml). A solution of L-valinol (0.99 g, 9.6mmol) in THF (40 ml) was added to give the title product (3.16 g); TLC,R_(f) =0.33, silica gel, EtOAc.

c. N-t-Butyloxycarbonyl-L-valyl-L-prolyl-L-valinal

A solution of the product of Example 37b (0.5 g, 0.78 mmol) in CH₂ Cl₂(1 ml) was added to a solution of pyridinium chlorochromate (0.445 g,2.2 mmol) in dry CH₂ Cl₂ (3 ml). The reaction mixture was stirred toroom temperature until the reaction was complete by TLC (R_(f) =0.8,silica gel, EtOAc). Dry Et₂ O was added to the reaction mixture and thesolution decanted. The black tarry residue was triturated with Et₂ O andthe solutions combined and filtered through a Florisil® column to givethe title product (0.100 g). (Florisil® is a registered trademark ofFloridin Co., Berkeley Springs, W. Va., U.S.A., for activated magnesiumsilicate in the form of hard porous stable white granules. This materialis useful as an analytical reagent, adsorbent and catalyst).

Elemental Analysis. Calculated for C₂₀ H₃₅ N₃ O₅.0.5H₂ O: C, 59.09; H,8.92; N, 10.33. Found: C, 59.13; H, 8.77; N, 9.98.

EXAMPLE 38 N-Succinyl-L-valyl-L-prolyl-L-valinal

Formula (III): R⁹ ═--CH(CH₃)₂ ; R¹⁰ ═--CH(CH₃)₂ ; R¹¹ ═--(CH₂)₂ COOH.

a. L-Valyl-L-prolyl-L-valinol

Trifluoroacetic acid (3.4 ml, 44 mmol) was added to a solution of theproduct of Example 37b (1.76 g, 4.4 mmol) in CH₂ Cl₂ (3.4 mmol) at roomtemperature and the mixture was stirred for 1 hr. The reaction mixturewas cooled to 0° C. and a solution of Na₂ CO₃ (2.6 g, 50 mmol) in H₂ O(10 ml) was added dropwise. The reaction mixture was diluted with CH₂Cl₂ and the organic layer separated, dried over Na₂ SO₄ and filtered togive the title product (0.93 g) as a hygroscopic solid.

b. N-Succinyl-L-valyl-L-prolyl-L-valinol

A solution of succinic anhydride (0.31 g, 3.1 mmol) in CH₃ CN was addedto a solution of the product of Example 38a (0.83 g, 2.8 mmol) in CH₃ CN(30 ml) at room temperature. The reaction mixture was stirred overnightand the solution concentrated under vacuum to give the crude product.Purification by flash chromatograpy on silica gel with MeOH:CHCl₃ :HOAc(4.5:95:0.5) gave the product (0.620 g) as a white solid; TLC, R_(f)=0.24, silica gel, MeOH:CHCl₃ :HOAc (4.5:95:0.5).

c. N-Succinyl-L-valyl-L-prolyl-L-valinal

A solution of DMSO (0.35 ml, 5.0 mmol) in CH₂ Cl₂ (1.0 ml) was added toa solution of oxalyl chloride (0.22 ml, 2.5 mmol) in CH₂ Cl₂ cooled to-23° C. under a nitrogen atmosphere. A solution of the product ofExample 38b (0.5 g, 1.25 mmol) in CH₂ Cl₂ (3 ml) was added dropwise. Thereaction mixture was stirred for 15 min. and TEA (0.69 ml, 5.0 mmol) wasadded dropwise. The reaction mixture was allowed to warm to roomtemperature and was diluted with CH₂ Cl₂. The solution was washed with10% aqueous AcOH and brine, dried over Na₂ SO₄, filtered andconcentrated to give the crude product (0.750 g) as a yellow oil. Theoil was purified by flash chromatography on silica gel with MeOH:CHCl₃:HOAc (4.5:95:0.5) to give the final product (0.042 g); TLC, R_(f)=0.33, silica gel, MeOH:CHCl.sub. 3 :HOAc (9.5:90:0.5).

EXAMPLE 39 N-Acetyl-L-valyl-L-prolyl-L-valinal

Formula (III): R⁹ ═--CH(CH₃)₂ ; R¹⁰ ═--CH(CH₃)₂ ; R¹¹ ═--CH₃ ;

a. N-Acetyl-L-valyl-L-prolyl-L-valinal diethylacetal

Acetyl chloride (0.41 g, 5.19 mmol) was added to a solution of theproduct of Example 13c (1.91 g, 5.19 mmol) in CH₂ Cl₂ (50 ml) andN-methylmorpholine (3 ml) at 0° C. under a nitrogen atmosphere. Thereaction mixture was stirred for 15 min. and then warmed to roomtemperature. Water (20 ml) was added. The organic layer was washedsuccessively with 20% citric acid, brine, saturated aqueous NaHCO₃ andbrine and dried over MgSO₄, filtered and concentrated to give a viscousoil. The product was purified by flash chromatography on silica gel withCH₂ Cl₂ :MeOH (98:2) to give an oil (1.3 g). TLC, R_(f) =0.5 silica gelCH₂ Cl₂ :MeOH (95:5).

Elemental Analysis. Calculated for C₂₁ H₃₉ N₃ O₅ : C, 60.99; H, 9.50; N,10.16. Found: C, 60.55; H, 9.42; N, 10.17.

b. N-Acetyl-L-valyl-L-prolyl-L-valinal

Dowex® 50WX-H resin (40 ml) was added to a solution of the product ofExample 39a (1.15 g, 2.78 mmol) in H₂ O (40 ml) and acetone (5 ml) atroom temperature under a nitrogen atmosphere and the mixture was stirredfor 20 hr. The mixture was filtered and the resin was washed withacetone. The filtrate and wash were combined and concentrated in vacuoto remove the acetone. Water was removed with lyophilizer to give theproduct as a white powder (0.757 g).

Elemental Analysis. Calculated for C₁₇ H₂₉ N₃ O₄.0.4H₂ O: C, 58.90; H,8.66; N, 12.10. Found: C, 58.93; H, 8.44; N, 11.97.

EXAMPLE 40 N-Benzyloxycarbonyl-L-valyl-L-prolyl-L-valinal

Formula III: R⁹ ═--CH(CH₃)₂ ; R¹⁰ ═--CH(CH₃)₂ ; R¹¹ ═--OCH₂ C₆ H₅

a. N-Benzyloxycarbonyl-L-valyl-L-prolyl-L-valinal

A mixture of p-toluenesulfonic acid (150 mg) and the product of Example13b (500 mg, 0.988 mmol) in acetone (70 ml) was stirred at roomtemperature for 3 hr. The mixture was concentrated under vacuum and theresidue was dissolved in EtOAc. This solution was washed with 5% aqueousNaHCO₃ and brine, dried over MgSO₄, filtered and concentrated to give aglass (410 mg); TLC, R_(f) =0.60, silica gel, CH₂ Cl₂ :MeOH (95:5).

b. N-Benzyloxycarbonyl-L-valyl-L-prolyl-L-valinal bisulfite adduct

The product of Example 40a was treated as described in Example 32h toprovide a white powder.

Elemental Analysis. Calculated for C₂₃ H₃₃ N₃ O₅.2NaHSO₃ : C, 43.18; H,5.52; N, 6.56. Found: C, 42.85; H, 5.64; N, 6.43.

What is claimed is:
 1. A compound of the following formula (I-A), (II-A)or (III-A): ##STR15## wherein R¹, R⁵ and R⁹ are lower alkyl groupscontaining from 3 to about 6 carbon atoms;R², R³, R⁶, R⁷ and R¹⁰ arealkyl groups of about 1 to 10 carbon atoms which may optionally besubstituted by a monocyclic aryl group or by an amide, urea or carbamategroup via the nitrogen thereof; and R⁴ and R¹¹ are lower alkyl,substituted lower alkyl, lower alkoxy or substituted lower alkoxy groupswherein the alkyl or alkoxy contains about 1 to 6 carbon atoms, ormonocyclic or bicyclic aryl groups; and R⁸ is hydroxy, a lower alkoxygroup containing about 1 to 6 carbon atoms, or an aralkoxy groupcontaining about 7 to 12 carbon atoms;and wherein --CHR² --, --CHR³ --,--CHR⁶ --, --CHR⁷ --, --CHR¹⁰ -- and the proline group are of theL-configuration.
 2. A compound of the following formula (I-B), (II-B),or (III-B): ##STR16## wherein R¹, R⁵ and R⁹ are lower alkyl groupscontaining from 3 to about 6 carbon atoms;R², R³, R⁶, R⁷ and R¹⁰ arealkyl groups of about 1 to 10 carbon atoms which may optionally besubstituted by a monocyclic aryl group or by an amide, urea or carbamategroup via the nitrogen thereof; and R⁴ and R¹¹ are lower alkyl,substituted lower alkyl, lower alkoxy or substituted lower alkoxy groupswherein the alkyl or alkoxy contains about 1 to 6 carbom atoms, ormonocyclic or bicyclic aryl groups; R⁸ is hydroxy, a lower groupcontaining about 1 to 6 carbon atoms, or an aralkoxy group containingabout 7 to 12 carbon atoms; and Q is an aldehyde protecting group;andwherein --CHR² --, --CHR³ --, --CHR⁶ --, --CHR⁷ --, --CHR¹⁰ -- and theproline group are of the L-configuration.
 3. A compound according toclaim 2 in which Q is (OR¹²)₂ where R¹² is a lower alkyl radicalcontaining from 1 to about 6 carbon atoms.